Patatin-like phospholipase domain containing 3 (PNPLA3) iRNA compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting the Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of a PNPLA3 gene and to methods of preventing and treating an PNPLA3-associated disorder, e.g., Nonalcoholic Fatty Liver Disease (NAFLD).

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2020/064776, filed on Dec. 14, 2020, which in turn claims the benefit of priority to U.S. Provisional Application No. 62/948,445, filed on Dec. 16, 2019, and U.S. Provisional Application No. 63/040,602, filed on Jun. 18, 2020. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 10, 2021 is named 121301_10603_SL.txt and is 1,097,214 bytes in size.

BACKGROUND OF THE INVENTION

The accumulation of excess triglyceride in the liver is known as hepatic steatosis (or fatty liver), and is associated with adverse metabolic consequences, including insulin resistance and dyslipidemia. Fatty liver is frequently found in subjects having excessive alcohol intake and subjects having obesity, diabetes, or hyperlipidemia. However, in the absence of excessive alcohol intake (>10 g/day), nonalcoholic fatty liver disease (NAFLD) can develop. NAFLD refers to a wide spectrum of liver diseases that can progress from simple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). All of the stages of NAFLD have in common the accumulation of fat (fatty infiltration) in the liver cells (hepatocytes).

The NAFLD spectrum begins with and progress from its simplest stage, called simple fatty liver (steatosis). Simple fatty liver involves the accumulation of fat (triglyceride) in the liver cells with no inflammation (hepatitis) or scarring (fibrosis). The next stage and degree of severity in the NAFLD spectrum is NASH, which involves the accumulation of fat in the liver cells, as well as inflammation of the liver. The inflammatory cells destroy liver cells (hepatocellular necrosis), and NASH ultimately leads to scarring of the liver (fibrosis), followed by irreversible, advanced scarring (cirrhosis). Cirrhosis that is caused by NASH is the last and most severe stage in the NAFLD spectrum.

In 2008, a genomewide association study of individuals with proton magnetic resonance spectroscopy of the liver to evaluate hepatic fat content, a significant association was identified between hepatic fat content and the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) gene (see, for example, Romeo et al. (2008) Nat. Genet., 40(12):1461-1465). Studies with knock-in mice have demonstrated that expression of a sequence polymorphism (rs738409, 1148M) in PNPLA3 causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of triglycerides in the liver (Smagris et al. (2015) Hepatology, 61:108-118). Specifically, the PNPLA31148M variant was associated with promoting the development of fibrogenesis by activating the hedgehog (Hh) signaling pathway, leading to the activation and proflieration of hepatic stellate cells and excessive generation and deposition of extracellular matrix (Chen et al. (2015) World J. Gastroenterol., 21(3):794-802).

Currently, treatments for NAFLD are directed towards weight loss and treatment of any secondary conditions, such as insulin resistance or dyslipidemia. To date, no pharmacologic treatments for NAFLD have been approved. Therefore, there is a need for therapies for subjects suffering from NAFLD.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3). The Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) may be within a cell, e.g., a cell within a subject, such as a human subject.

In an aspect, the invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2. In one embodiment, the dsRNA agent comprises at least one thermally destabilizing nucleotide modification, e.g., an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2′-deoxy modification, an acyclic nucleotide, an unlocked nucleic acids (UNA), or a glycerol nucleic acid (GNA), e.g., the antisense strand comprises at least one thermally destabilizing nucleotide modification.

In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), and wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequence of nucleotides 677-721; 683-721; 773-817; 1185-1295; 1185-1241; 1202-1295; 1202-1241; 1255-1295; 1738-1792; 1901-1945; 1920-1945; 2108-2208; 2108-2166; 2108-2136, 2121-2166; 2121-2208; 2169-2208; 2176-2208; or 2239-2265 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 19 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequence of nucleotides 574-596; 677-699; 683-705; 699-721; 773-795; 795-817; 1185-1207; 1192-1214; 1202-1224; 1208-1230; 1209-1231; 1210-1232; 1211-1233; 1212-1234; 1213-1233; 1214-1234; 1214-1236; 1215-1237; 1216-1238; 1219-1237; 1219-1241; 1255-1275; 1256-1276; 1257-1275; 1257-1277; 1258-1278; 1259-1279; 1260-1278; 1260-1280; 1261-1281; 1262-1282; 1263-1283; 1264-1282; 1264-1284; 1265-1285; 1267-1285; 1266-1286; 1266-1288; 1267-1285; 1267-1287; 1268-1290; 1269-1289; 1270-1290; 1271-1291; 1272-1292; 1273-1293; 1274-1294; 1275-1295; 1631-1653; 1738-1760; 1739-1761; 1740-1760; 1740-1762; 1741-1763; 1744-1766; 1746-1766; 1750-1772; 1751-1773; 1752-1774; 1753-1775; 1754-1776; 1755-1777; 1756-1778; 1757-1779; 1758-1780; 1759-1781; 1760-1782; 1761-1783; 1762-1782; 1762-1784; 1763-1785; 1764-1786; 1765-1787; 1766-1786; 1766-1788; 1767-1787; 1768-1788; 1767-1789; 1769-1789; 1770-1788; 1770-1790; 1771-1791; 1772-1792; 1815-1837; 1901-1923, 1920-1942, 1923-1945; 2112-2130; 2169-2191; 2171-2191; 2176-2198, 2177-2199, 2178-2200; 2179-2201, 2180-2202; 2181-2203; 2183-2205; 2184-2206; 2186-2208; 2239-2261; 2241-2263; 2242-2264; or 2243-2265 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 19 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by nor more than 0, 1, 2, or 3 nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-517197.2; AD-517258.2; AD-516748.2; AD-516851.2; AD-519351.2; AD-519754.2; AD-519828.2; AD-520018.2; AD-520035.2; AD-520062.2; AD-520064.2; AD-520065.2; AD-520067.2; AD-75289.2; AD-520069.2; AD-520099.2; AD-67575.7; AD-520101.2; AD-67605.7; AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; and AD-1193481.1.

In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by nor more than 0, 1, 2, or 3 nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1, AD-519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2, AD-520063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516828.2, AD-518983.2, AD-805636.2, AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2, AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2; AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; and AD-1193481.1.

In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides differing by nor more than 0, 1, 2, or 3 nucleotides from any one of the antisense strand nucleotide sequences of duplex AD-519351.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In one embodiment, substantially all of the nucleotides of the sense strand; substantially all of the nucleotides of the antisense strand comprise a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, all of the nucleotides of the sense strand comprise a modification; all of the nucleotides of the antisense strand comprise a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.

In one embodiment, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and glycol; and combinations thereof.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, and, a vinyl-phosphonate nucleotide; and combinations thereof.

In another embodiment, at least one of the modifications on the nucleotides is a thermally destabilizing nucleotide modification.

In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2′-deoxy modification, an acyclic nucleotide, an unlocked nucleic acids (UNA), and a glycerol nucleic acid (GNA).

In some embodiments, the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In some embodiments, the modifications on the nucleotides are 2′-O-methyl, GNA and 2′fluoro modifications.

In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

The double stranded region may be 19-30 nucleotide pairs in length; 19-25 nucleotide pairs in length; 19-23 nucleotide pairs in length; 23-27 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

In one embodiment, each strand is independently no more than 30 nucleotides in length.

In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

The region of complementarity may be at least 17 nucleotides in length; between 19 and 23 nucleotides in length; or 19 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.

In one embodiment, the ligand is

In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand, e.g., the antisense strand or the sense strand.

In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand, e.g., the antisense strand or the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. In one embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

The present invention also provides cells containing any of the dsRNA agents of the invention and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

The pharmaceutical composition of the invention may include dsRNA agent in an unbuffered solution, e.g., saline or water, or the pharmaceutical composition of the invention may include the dsRNA agent is in a buffer solution, e.g., a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof; or phosphate buffered saline (PBS).

In one aspect, the present invention provides a method of inhibiting expression of a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene in a cell. The method includes contacting the cell with any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby inhibiting expression of the PNPLA3 gene in the cell.

In one embodiment, the cell is within a subject, e.g., a human subject, e.g., a subject having a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disorder, such as a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disorder selected from the group consisting of fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

In one embodiment, contacting the cell with the dsRNA agent inhibits the expression of PNPLA3 by at least 50%, 60%, 70%, 80%, 90%, or 95%.

In one embodiment, inhibiting expression of PNPLA3 decreases PNPLA3 protein level in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

In one aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduction in Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) expression. The method includes administering to the subject a therapeutically effective amount of any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby treating the subject having the disorder that would benefit from reduction in PNPLA3 expression.

In another aspect, the present invention provides a method of preventing at least one symptom in a subject having a disorder that would benefit from reduction in Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) expression. The method includes administering to the subject a prophylactically effective amount of any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduction in PNPLA3 expression.

In one embodiment, the disorder is a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disorder, e.g., a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disorder is selected from the group consisting of fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

In one embodiment, the PNPLA3-associated disorder is NAFLD.

In one embodiment, the subject is human.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the methods of the invention include further determining the level of PNPLA3 in a sample(s) from the subject.

In one embodiment, the level of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in the subject sample(s) is a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) protein level in a blood or serum sample(s).

In certain embodiments, the methods of the invention further comprise administering to the subject an additional therapeutic agent. In a further embodiment, the additional therapeutic agent is selected from the group consisting of an HMG-CoA reductase inhibitor, a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant, a glycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor, a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or fish oil.

The present invention also provides kits comprising any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, and optionally, instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 3 mg/kg or 10 mg/kg dose of the indicated dsRNA duplexes, on day 7 or day 14 post-dose. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 2 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single dose of the indicated dsRNA duplexes, on day 7 post-dose as described in Example 3. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 3 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 10 mg/kg dose of the indicated dsRNA duplexes, on day 7 post-dose. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 4 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 10 mg/kg dose or multiple 10 mg/kg doses of the indicated dsRNA duplexes, on day 7 post-dose as described in Example 3. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 5 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 10 mg/kg dose of the indicated dsRNA duplexes, on day 7 post-dose. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 6 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 10 mg/kg dose of the indicated dsRNA duplexes, on day 7 post-dose. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

FIG. 7 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) subcutaneously administered a single 10 mg/kg dose of the indicated dsRNA duplexes, on day 7 post-dose. Human PNPLA3 mRNA levels are shown relative to control levels detected with PBS treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene) in mammals.

The iRNAs of the invention have been designed to target the human Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene, including portions of the gene that are conserved in the Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) orthologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites or the specific modifications in these iRNAs confer to the iRNAs of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention provides methods for treating and preventing an Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disorder, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD), using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PNPLA3 gene.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an PNPLA3 gene.

In certain embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an PNPLA3 gene. In some embodiments, such iRNA agents having longer length antisense strands preferably may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation of mRNAs of the corresponding gene (PNPLA3 gene) in mammals. Using in vitro assays, the present inventors have demonstrated that iRNAs targeting a PNPLA3 gene can potently mediate RNAi, resulting in significant inhibition of expression of a PNPLA3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject having a PNPLA3-associated disorder, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

Accordingly, the present invention provides methods and combination therapies for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an PNPLA3 gene, e.g., a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3)-associated disease, such as fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD), using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an PNPLA3 gene.

The present invention also provides methods for preventing at least one symptom in a subject having a disorder that would benefit from inhibiting or reducing the expression of a PNPLA3 gene, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). For example, in a subject having NAFLLD, the methods of the present invention may reduce at least one symptom in the subject, e.g., fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider-like blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid build up and swelling of the legs (edema), abdomen swelling (ascites), and mental confusion.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a PNPLA3 gene as well as compositions, uses, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of a PNPLA3 gene, e.g., subjects susceptible to or diagnosed with a PNPLA3-associated disorder.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

As used herein, “Patatin-Like Phospholipase Domain Containing 3,” used interchangeably with the term “PNPLA3,” refers to the well-known gene that encodes a triacylglycerol lipase that mediates triacyl glycerol hydrolysis in adipocytes.

Exemplary nucleotide and amino acid sequences of PNPLA3 can be found, for example, at GenBank Accession No. NM_025225.2 (Homo sapiens PNPLA3; SEQ ID NO:1; reverse complement, SEQ ID NO:2); GenBank Accession No. NM_054088.3 (Mus musculus PNPLA3; SEQ ID NO:3; reverse complement, SEQ ID NO:4); GenBank Accession No. NM_001282324.1 (Rattus norvegicus PNPLA3; SEQ ID NO:5; reverse complement, SEQ ID NO:6); GenBank Accession No. XM_005567051.1 (Macaca fascicularis PNPLA3, SEQ ID NO:7; reverse complement, SEQ ID NO:8); GenBank Accession No. XM_001109144.2 (Macaca mulatta PNPLA3, SEQ ID NO:9; reverse complement, SEQ ID NO:10); and GenBank Accession No. XM_005567052.1 (Macaca fascicularis PNPLA3, SEQ ID NO:11; reverse complement, SEQ ID NO:12).

Additional examples of PNPLA3 mRNA sequences are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

Further information on PNPLA3 can be found, for example, at www.ncbi.nlm.nih.gov/gene/?term=pnpla3.

The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

The term PNPLA3, as used herein, also refers to variations of the PNPLA3 gene including variants provided in the SNP database. Numerous sequence variations within the PNPLA3 gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov/snp/?term=pnpla3, the entire contents of which is incorporated herein by reference as of the date of filing this application.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a PNPLA3 gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a PNPLA3 gene. In one embodiment, the target sequence is within the protein coding region of PNPLA3.

The target sequence may be from about 19-36 nucleotides in length, e.g., preferably about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of a PNPLA3 gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., a PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a PNPLA3 gene. Accordingly, the term “siRNA” is also used herein to refer to an iRNA as described above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNA agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a PNPLA3 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide. In addition, as used in this specification, an “iRNA” may include ribonucleotides with chemical modifications; an iRNA may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide if present within an RNAi agent can be considered to constitute a modified nucleotide.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not be, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a PNPLA3 gene, to direct cleavage of the target RNA.

In some embodiments, an iRNA of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., a PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double stranded iRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNA agent, i.e., no nucleotide overhang. A “blunt ended” double stranded RNA agent is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleotide overhangs at either end. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a PNPLA3 mRNA.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a PNPLA3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand double stranded RNA agent of the invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region.

Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a PNPLA3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a PNPLA3 gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a PNPLA3 gene is important, especially if the particular region of complementarity in a PNPLA3 gene is known to have polymorphic sequence variation within the population.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a double stranded RNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a PNPLA3 gene). For example, a polynucleotide is complementary to at least a part of a PNPLA3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a PNPLA3 gene.

Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target PNPLA3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, 9, or 11, or a fragment of any one of SEQ ID NOs:1, 3, 5, 7, 9, or 11, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to a fragment of SEQ ID NO: 1 selected from the group of nucleotides 677-721; 683-721; 773-817; 1185-1295; 1185-1241; 1202-1295; 1202-1241; 1255-1295; 1738-1792; 1901-1945; 1920-1945; 2108-2208; 2108-2166; 2108-2136, 2121-2166; 2121-2208; 2169-2208; 2176-2208; or 2239-2265 of SEQ ID NO: 1, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to a fragment of SEQ ID NO: 1 selected from the group of nucleotides 574-596; 677-699; 683-705; 699-721; 773-795; 795-817; 1185-1207; 1192-1214; 1202-1224; 1208-1230; 1209-1231; 1210-1232; 1211-1233; 1212-1234; 1213-1233; 1214-1234; 1214-1236; 1215-1237; 1216-1238; 1219-1237; 1219-1241; 1255-1275; 1256-1276; 1257-1275; 1257-1277; 1258-1278; 1259-1279; 1260-1278; 1260-1280; 1261-1281; 1262-1282; 1263-1283; 1264-1282; 1264-1284; 1265-1285; 1267-1285; 1266-1286; 1266-1288; 1267-1285; 1267-1287; 1268-1290; 1269-1289; 1270-1290; 1271-1291; 1272-1292; 1273-1293; 1274-1294; 1275-1295; 1631-1653; 1738-1760; 1739-1761; 1740-1760; 1740-1762; 1741-1763; 1744-1766; 1746-1766; 1750-1772; 1751-1773; 1752-1774; 1753-1775; 1754-1776; 1755-1777; 1756-1778; 1757-1779; 1758-1780; 1759-1781; 1760-1782; 1761-1783; 1762-1782; 1762-1784; 1763-1785; 1764-1786; 1765-1787; 1766-1786; 1766-1788; 1767-1787; 1768-1788; 1767-1789; 1769-1789; 1770-1788; 1770-1790; 1771-1791; 1772-1792; 1815-1837; 1901-1923, 1920-1942, 1923-1945; 2112-2130; 2169-2191; 2171-2191; 2176-2198, 2177-2199, 2178-2200; 2179-2201, 2180-2202; 2181-2203; 2183-2205; 2184-2206; 2186-2208; 2239-2261; 2241-2263; 2242-2264; or 2243-2265 of SEQ ID NO: 1, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, or 12, or a fragment of any one of SEQ ID NOs:2, 4, 6, 8, 10, or 12, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

In some embodiments, an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

In certain embodiments, the sense and antisense strands are selected from any one of duplexes AD-517197.2; AD-517258.2; AD-516748.2; AD-516851.2; AD-519351.2; AD-519754.2; AD-519828.2; AD-520018.2; AD-520035.2; AD-520062.2; AD-520064.2; AD-520065.2; AD-520067.2; AD-75289.2; AD-520069.2; AD-520099.2; AD-67575.7; AD-520101.2; AD-67605.7, AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; and AD-1193481.1 In some embodiments, the sense and antisense strands are selected from any one of duplexes AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1, AD-519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2, AD-520063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516828.2, AD-518983.2, AD-805636.2, AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2, AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2, AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; and AD-1193481.1.

In some embodiments, the sense and antisense strands are from duplex AD-519351.

In general, an “iRNA” includes ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a dsRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide if present within an RNAi agent can be considered to constitute a modified nucleotide.

In an aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the iRNA may be put into physical contact with the cell by the individual performing the method, or alternatively, the iRNA may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA. Contacting a cell in vivo may be done, for example, by injecting the iRNA into or near the tissue where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the iRNA may contain or be coupled to a ligand, e.g., GalNAc, that directs the iRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an iRNA and subsequently transplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease or disorder that would benefit from reduction in PNPLA3 expression; a human at risk for a disease or disorder that would benefit from reduction in PNPLA3 expression; a human having a disease or disorder that would benefit from reduction in PNPLA3 expression; or human being treated for a disease or disorder that would benefit from reduction in PNPLA3 expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result, such as reducing at least one sign or symptom of a PNPLA3-associated disorder in a subject. Treatment also includes a reduction of one or more sign or symptoms associated with unwanted PNPLA3 expression; diminishing the extent of unwanted PNPLA3 activation or stabilization; amelioration or palliation of unwanted PNPLA3 activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment. The term “lower” in the context of the level of PNPLA3 in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of PNPLA3 in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, “lower” is the decrease in the difference between the level of a marker or symptom for a subject suffering from a disease and a level accepted within the range of normal for an individual, e.g., the level of decrease in bodyweight between an obese individual and an individual having a weight accepted within the range of normal.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, may be treated or ameliorated by a reduction in expression of an PNPLA3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of unwanted or excessive PNPLA3 expression, such as the presence of elevated levels of proteins in the hedgehog signaling pathway, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The likelihood of developing, e.g., NAFLD, is reduced, for example, when an individual having one or more risk factors for NAFLD either fails to develop NAFLD or develops NAFLD with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “Patatin-Like Phospholipase Domain Containing 3-associated disease” or “PNPLA3-associated disease,” is a disease or disorder that is caused by, or associated with PNPLA3 gene expression or PNPLA3 protein production. The term “PNPLA3-associated disease” includes a disease, disorder or condition that would benefit from a decrease in PNPLA3 gene expression, replication, or protein activity. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic steatohepatitis (NASH). In another embodiment, the PNPLA3-associated disease is liver cirrhosis. In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance. In one embodiment, the PNPLA3-associated disease is obesity.

In one embodiment, a PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). As used herein, “nonalcoholic fatty liver disease,” used interchangeably with the term “NAFLD,” refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol ingestion per day. NAFLD is the most common liver disease in the United States, and is commonly associated with insulin resistance/type 2 diabetes mellitus and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which are incorporated herein by reference.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a PNPLA3-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a PNPLA3-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment. The iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to urine obtained from the subject. A “sample derived from a subject” can refer to blood or blood derived serum or plasma from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of a PNPLA3 gene. In preferred embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an PNPLA3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human susceptible to developing a PNPLA3-associated disorder, e.g., hypertriglyceridemia. The dsRNAi agent includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a PNPLA3 gene. The region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). Upon contact with a cell expressing the PNPLA3 gene, the iRNA inhibits the expression of the PNPLA3 gene (e.g., a human, a primate, a non-primate, or a rat PNPLA3 gene) by at least about 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flow cytometric techniques. In preferred embodiments, inhibition of expression is determined by the qPCR method provided in the examples herein with the siRNA at, e.g., a 10 nM concentration, in an appropriate organism cell line provided therein. In preferred embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e.g., a mouse or an AAV-infected mouse expressing the human target gene, e.g., when administered as single dose, e.g., at 3 mg/kg at the nadir of RNA expression.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a PNPLA3 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target PNPLA3 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art. Double stranded RNAi compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a PNPLA3 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50.

In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

In certain embodiments, the sense or antisense strand is selected from the sense or antisense strand of any one of duplexes AD-517197.2; AD-517258.2; AD-516748.2; AD-516851.2; AD-519351.2; AD-519754.2; AD-519828.2; AD-520018.2; AD-520035.2; AD-520062.2; AD-520064.2; AD-520065.2; AD-520067.2; AD-75289.2; AD-520069.2; AD-520099.2; AD-67575.7; AD-520101.2; AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; AD-1193481.1 or AD-67605.7.

In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of any one of duplexes AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1, AD-519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2, AD-520063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516828.2, AD-518983.2, AD-805636.2, AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2, AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2, AD-1193323.1; AD-1193344.1; AD-1193350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; or AD-1193481.1.

In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of duplex AD-519351.

It will be understood that, although the sequences in, for example, Tables 3, 5, 7, 9, 11, 21, 24, 27, 30, 32, 36 and 50 are not described as modified or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. In other words, the invention encompasses dsRNA of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50 which are un-modified, un-conjugated, modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50. dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences of any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50, and differing in their ability to inhibit the expression of a PNPLA3 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50 identify a site(s) in a PNPLA3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a PNPLA3 gene.

III. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA or substantially all of the nucleotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the iRNA.

The nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments of the invention, the dsRNA agents of the invention are in a free acid form. In other embodiments of the invention, the dsRNA agents of the invention are in a salt form. In one embodiment, the dsRNA agents of the invention are in a sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothiotate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages without a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.

Representative U.S. Patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂ component parts.

Representative U.S. Patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound in which an RNA mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative US patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the RNA of an iRNA can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and U.S. Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, U.S. Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and U.S. Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an iRNA. Suitable phosphate mimics are disclosed in, for example U.S. Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNA agents of the invention include agents with chemical modifications as disclosed, for example, in WO2013/075035, the entire contents of each of which are incorporated herein by reference. WO2013/075035 provides motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of a dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The dsRNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of the double stranded RNA agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNAi agent, the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNA agents capable of inhibiting the expression of a target gene (i.e., PNPLA3 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” The duplex region of a dsRNAi agent may be, for example, the duplex region can be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be, independently, 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, the overhang regions can include extended overhang regions as provided above. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3′-overhang is present in the antisense strand. In some embodiments, this 3′-overhang is present in the sense strand.

The dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-end of the sense strand or, alternatively, at the 3-end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In certain embodiments, every nucleotide in the sense strand and the antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In certain embodiments each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In certain embodiments, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein Dicer cleavage of the dsRNAi agent preferentially results in an siRNA comprising the 3′-end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 19-23 nucleotides in length, the cleavage site of the antisense strand is typically around the 10, 11, and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; the 10, 11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; or the 13, 14, 15 positions of the antisense strand, the count starting from the first nucleotide from the 5′-end of the antisense strand, or, the count starting from the first paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistries of the motifs are distinct from each other, and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end, or both ends of the strand.

In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two, or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two, or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′- or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′- or 5′-overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications.

Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′ to 3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand or antisense strand interrupts the initial modification pattern present in the sense strand or antisense strand. This interruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense or antisense strand may enhance the gene silencing activity against the target gene.

In some embodiments, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . NaYYYN_(b) . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_(a)” and “N_(b)” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_(a) and N_(b) can be the same or different modifications. Alternatively, N_(a) or N_(b) may be present or absent when there is a wing modification present.

The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand, antisense strand, or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-end and two phosphorothioate internucleotide linkages at the 3′-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, or the 5′end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In certain embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In other embodiments, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). For example, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented by formula (I): 5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sense strand, the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas: 5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Ib); 5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′  (Ic); or 5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5, or 6 Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula: 5′n _(p)-N_(a)—YYY—N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II): 5′n _(q)′—N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′X′X′X′)_(i)—N′_(a)-n _(p)′3′  (II)

wherein:

k and 1 are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas: 5′n _(q)′—N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n _(p),3′  (IIb); 5′n _(q)′—N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n _(p),3′  (IIc); or 5′n _(q)′—N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n _(p)′3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula: 5′n _(p′)—N_(a′)—Y′Y′Y′—N_(a)′-n _(q)′3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may contain YYY motif occurring at 9, 10, and 11 positions of the strand when the duplex region is 21 nt, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the dsRNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the iRNA duplex represented by formula (III): sense: 5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′ antisense: 3′n _(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(i)—N_(a)′-n _(q)′5′   (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming an iRNA duplex include the formulas below: 5′n _(p)-N_(a)—YYY—N_(a)-n _(q)3′ 3′n _(p)′—N_(a)′—Y′Y′Y′—N_(a) ′n _(q)′5′   (IIIa) 5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′ 3′n _(p)′—N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a) ′n _(q)′5′   (IIIb) 5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′ 3′n _(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n _(q)′5′   (IIIc) 5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′ 3′n _(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n _(q)′5′   (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b), and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends, and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

In certain embodiments, an RNAi agent of the invention may contain a low number of nucleotides containing a 2′-fluoro modification, e.g., 10 or fewer nucleotides with 2′-fluoro modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent of the invention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 6 nucleotides with a 2′-fluoro modification in the antisense strand. In another specific embodiment, the RNAi agent of the invention contains 6 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain an ultra low number of nucleotides containing a 2′-fluoro modification, e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. For example, the RNAi agent may contain 2, 1 of 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent may contain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotides with a 2-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

Various publications describe multimeric iRNAs that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:

A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:

As described in more detail below, the iRNA that contains conjugations of one or more carbohydrate moieties to an iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of a iRNA can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or diethanolamine backbone.

i. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5′-end of the antisense strand.

An iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent may be represented by formula (L):

In formula (L), B1, B2, B3, B1′, B2′, B3′, and B4′ each are independently a nucleotide containing a modification selected from the group consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe modifications. In one embodiment, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-F modifications. In one embodiment, at least one of B1, B2, B3, B1′, B2′, B3′, and B4′ contain 2′-O—N-methylacetamido (2′-O-NMA) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5′-end of the antisense strand). For example, C1 is at a position of the sense strand that pairs with a nucleotide at positions 2-8 of the 5′-end of the antisense strand. In one example, C1 is at position 15 from the 5′-end of the sense strand. C1 nucleotide bears the thermally destabilizing modification which can include abasic modification; mismatch with the opposing nucleotide in the duplex; and sugar modification such as 2′-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 has thermally destabilizing modification selected from the group consisting of: i) mismatch with the opposing nucleotide in the antisense strand; ii) abasic modification selected from the group consisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2′-deoxynucleobase. In one example, the thermally destabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less or equal to the steric bulk of a 2′-OMe modification. A steric bulk refers to the sum of steric effects of a modification. Methods for determining steric effects of a modification of a nucleotide are known to one skilled in the art. The modification can be at the 2′ position of a ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2′ position of the ribose sugar, and provides the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′ are each independently selected from DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl. In one embodiment, T1 is DNA. In one embodiment, T1′ is DNA, RNA or LNA. In one embodiment, T2′ is DNA or RNA. In one embodiment, T3′ is DNA or RNA. n¹, n³, and q¹ are independently 4 to 15 nucleotides in length. n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length. n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length; alternatively, n⁴ is 0. q⁵ is independently 0-10 nucleotide(s) in length. n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶ are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴, q², q⁴, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n⁴ is 1. In one embodiment, C1 is at position 15 of the 5′-end of the sense strand

In one embodiment, T3′ starts at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q² is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ end of the antisense strand and T1′ starts from position 14 from the 5′ end of the antisense strand. In one example, T3′ starts from position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1 and T1′ starts from position 14 from the 5′ end of the antisense strand and q² is equal to 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length (i.e. not counting the T1′ and T3′ nucleotides).

In one embodiment, T1′ is at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q² is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1,

In one embodiment, T2′ starts at position 6 from the 5′ end of the antisense strand. In one example, T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴ is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sense strand, for instance, at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1; T1′ is at position 14 from the 5′ end of the antisense strand, and q² is equal to 1, and the modification to T1′ is at the 2′ position of a ribose sugar or at positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴ is 1; and T3′ is at position 2 from the 5′ end of the antisense strand, and q⁶ is equal to 1, and the modification to T3′ is at the 2′ position or at positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T2′ starts at position 8 from the 5′ end of the antisense strand. In one example, T2′ starts at position 8 from the 5′ end of the antisense strand, and q⁴ is 2.

In one embodiment, T2′ starts at position 9 from the 5′ end of the antisense strand. In one example, T2′ is at position 9 from the 5′ end of the antisense strand, and q⁴ is 1.

In one embodiment, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

The RNAi agent can comprise a phosphorus-containing group at the 5′-end of the sense strand or antisense strand. The 5′-end phosphorus-containing group can be 5′-end phosphate (5′-P), 5′-end phosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-end vinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or 5′-deoxy-5′-C-malonyl

When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate (5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e., trans-vinylphosphate,

5′-Z-VP isomer (i.e., cis-vinylphosphate,

or mixtures thereof. In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the sense strand. In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-P. In one embodiment, the RNAi agent comprises a 5′-P in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS. In one embodiment, the RNAi agent comprises a 5′-PS in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-VP. In one embodiment, the RNAi agent comprises a 5′-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5′-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5′-Z-VP in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment, the RNAi agent comprises a 5′-PS₂ in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment, the RNAi agent comprises a 5′-deoxy-5′-C-malonyl in the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The dsRNAi RNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof), and a targeting ligand.

In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In a particular embodiment, an RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker; and         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             13, 17, 19, and 21, and 2′-OMe modifications at positions 2,             4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5′             end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13,             15, 17, 19, 21, and 23, and 2′F modifications at positions             2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the             5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 21 and 22, and between nucleotide             positions 22 and 23 (counting from the 5′ end);     -   wherein the dsRNA agents have a two nucleotide overhang at the         3′-end of the antisense strand, and a blunt end at the 5′-end of         the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             13, 15, 17, 19, and 21, and 2′-OMe modifications at             positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to             13, 15, 17, 19, and 21 to 23, and 2′F modifications at             positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and             12 to 21, 2′-F modifications at positions 7, and 9, and a             deoxy-nucleotide (e.g. dT) at position 11 (counting from the             5′ end); and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 7, 9, 11, 13,             15, 17, and 19 to 23, and 2′-F modifications at positions 2,             4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5′             end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12,             14, and 16 to 21, and 2′-F modifications at positions 7, 9,             11, 13, and 15; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 5, 7, 9, 11, 13,             15, 17, 19, and 21 to 23, and 2′-F modifications at             positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting             from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 9, and 12 to             21, and 2′-F modifications at positions 10, and 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to             13, 15, 17, 19, and 21 to 23, and 2′-F modifications at             positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             and 13, and 2′-OMe modifications at positions 2, 4, 6, 8,             12, and 14 to 21; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11             to 13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at             positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′             end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12,             14, 15, 17, and 19 to 21, and 2′-F modifications at             positions 3, 5, 7, 9 to 11, 13, 16, and 18; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 25 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to             13, 15, 17, and 19 to 23, 2′-F modifications at positions 2,             3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g.             dT) at positions 24 and 25 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a four nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to             21, and 2′-F modifications at positions 7, and 9 to 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10             to 13, 15, and 17 to 23, and 2′-F modifications at positions             2, 6, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to             21, and 2′-F modifications at positions 7, and 9 to 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to             13, 15, and 17 to 23, and 2′-F modifications at positions 2,             6, 8, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 19 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to             19, and 2′-F modifications at positions 5, and 7 to 9; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end);     -   and     -   (b) an antisense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to             13, 15, and 17 to 21, and 2′-F modifications at positions 2,             6, 8, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 19 and 20, and between             nucleotide positions 20 and 21 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from agents listed in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49 or 50. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556). In other embodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting, or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands do not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other methods for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In other embodiments, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:15) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:16) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:17) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments the GalNAc conjugate is conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

Formula XXXIV.

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXXVI), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one or more GalNAc or GalNAc derivative attached to the iRNA agent. The GalNAc may be attached to any nucleotide via a linker on the sense strand or antsisense strand. The GalNac may be attached to the 5′-end of the sense strand, the 3′ end of the sense strand, the 5′-end of the antisense strand, or the 3′-end of the antisense strand. In one embodiment, the GalNAc is attached to the 3′ end of the sense strand, e.g., via a trivalent linker.

In other embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of linkers, e.g., monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention is part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O; Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O); R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)CHR^(a))—NH—, CO, CH═N—O,

or heterocyclyl; L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XLIX):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. Patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAi agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject susceptible to or diagnosed with a PNPLA3-associated disorder, e.g., NAFLD) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602). Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H, et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N, et al (2003), supra), “solid nucleic acid lipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the PNPLA3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for preventing or treating a PNPLA3-associated disorder, e.g., hypertriglyceridemia. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PNPLA3 gene.

In some embodiments, the pharmaceutical compositions of the invention are sterile. In another embodiment, the pharmaceutical compositions of the invention are pyrogen free.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PNPLA3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every month, once every 3-6 months, or once a year. In certain embodiments, the iRNA is administered about once per month to about once per six months.

After an initial treatment regimen, the treatments can be administered on a less frequent basis. Duration of treatment can be determined based on the severity of disease.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that doses are administered at not more than 1, 2, 3, or 4 month intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered about once per month. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered quarterly (i.e., about every three months). In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered twice per year (i.e., about once every six months).

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to mutations present in the subject, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a prophylactically or therapeutically effective amount, as appropriate, of a composition can include a single treatment or a series of treatments.

The iRNA can be delivered in a manner to target a particular tissue (e.g., hepatocytes).

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers.

A. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution either in the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

The application of emulsion formulations via dermatological, oral, and parenteral routes, and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers and their use in manufacture of pharmaceutical compositions and delivery of pharmaceutical agents are well known in the art.

v. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Such agent are well known in the art.

vi. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, or aromatic substances, and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating a PNPLA33-associated disorder, e.g., NAFLD.

Toxicity and prophylactic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose prophylactically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50, preferably an ED80 or ED90, with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the prophylactically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) or higher levels of inhibition as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents used for the prevention or treatment of aPNPLA3-associated disorder, e.g., NAFLD. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods for Inhibiting PNPLA3 Expression

The present invention also provides methods of inhibiting expression of a PNPLA3 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNA agent, in an amount effective to inhibit expression of PNPLA3 in the cell, thereby inhibiting expression of PNPLA3 in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNA agent, may be done in vitro or in vivo. Contacting a cell in vivo with the iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the iRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a PNPLA3” is intended to refer to inhibition of expression of any PNPLA3 gene (such as, e.g., a mouse PNPLA33 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene) as well as variants or mutants of a PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene, or a transgenic PNPLA3 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a PNPLA3 gene” includes any level of inhibition of aPNPLA3 gene, e.g., at least partial suppression of the expression of a PNPLA3 gene. The expression of the PNPLA3 gene may be assessed based on the level, or the change in the level, of any variable associated with PNPLA3 gene expression, e.g., PNPLA3 mRNA level or PNPLA3 protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject. It is understood that PNPLA3 is expressed predominantly in the liver, but also in the brain, gall bladder, heart, and kidney, and is present in circulation.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with PNPLA3 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of a PNPLA3 gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In preferred embodiments, expression of a PNPLA3 gene is inhibited by at least 70%. It is further understood that inhibition of PNPLA3 expression in certain tissues, e.g., in liver, without a significant inhibition of expression in other tissues, e.g., brain, may be desirable. In preferred embodiments, expression level is determined using the assay method provided in Example 2 with a 10 nM siRNA concentration in the appropriate species matched cell line.

In certain embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e.g., an AAV-infected mouse expressing the human target gene (i.e., PNPLA3), e.g., when administered as a single dose, e.g., at 3 mg/kg at the nadir of RNA expression. Knockdown of expression of an endogenous gene in a model animal system can also be determined, e.g., after administration of a single dose at, e.g., 3 mg/kg at the nadir of RNA expression. Such systems are useful when the nucleic acid sequence of the human gene and the model animal gene are sufficiently close such that the human iRNA provides effective knockdown of the model animal gene. RNA expression in liver is determined using the PCR methods provided in Example 2.

Inhibition of the expression of a PNPLA3 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a PNPLA3 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an iRNA of the invention, or by administering an iRNA of the invention to a subject in which the cells are or were present) such that the expression of a PNPLA3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an iRNA or not treated with an iRNA targeted to the gene of interest). In preferred embodiments, the inhibition is assessed by the method provided in Example 2 using a 10 nM siRNA concentration in the species matched cell line and expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a PNPLA3 gene may be assessed in terms of a reduction of a parameter that is functionally linked to PNPLA3 gene expression, e.g., PNPLA3 protein level in blood or serum from a subject. PNPLA3 gene silencing may be determined in any cell expressing PNPLA3, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of a PNPLA3 protein may be manifested by a reduction in the level of the PNPLA3 protein that is expressed by a cell or group of cells or in a subject sample (e.g., the level of protein in a blood sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells, or the change in the level of protein in a subject sample, e.g., blood or serum derived therefrom.

A control cell, a group of cells, or subject sample that may be used to assess the inhibition of the expression of a PNPLA3 gene includes a cell, group of cells, or subject sample that has not yet been contacted with an RNAi agent of the invention. For example, the control cell, group of cells, or subject sample may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent or an appropriately matched population control.

The level of PNPLA3 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of PNPLA3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the PNPLA3 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene™ (PreAnalytix™, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis.

In some embodiments, the level of expression of PNPLA3 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific PNPLA3. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of PNPLA3 mRNA.

An alternative method for determining the level of expression of PNPLA3 in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of PNPLA3 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System). In preferred embodiments, expression level is determined by the method provided in Example 2 using, e.g., a 10 nM siRNA concentration, in the species matched cell line.

The expression levels of PNPLA3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of PNPLA3 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein. In preferred embodiments, expression level is determined by the method provided in Example 2 using a 10 nM siRNA concentration in the species matched cell line.

The level of PNPLA3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention are assessed by a decrease in PNPLA3 mRNA or protein level (e.g., in a liver biopsy).

In some embodiments of the methods of the invention, the iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. The inhibition of expression of PNPLA3 may be assessed using measurements of the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from fluid or tissue from the specific site within the subject (e.g., liver or blood).

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

VII. Prophylactic and Treatment Methods of the Invention

The present invention also provides methods of using an iRNA of the invention or a composition containing an iRNA of the invention to inhibit expression of PNPLA3, thereby preventing or treating an PNPLA3-associated disorder, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In the methods of the invention the cell may be contacted with the siRNA in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an PNPLA3 gene, e.g., a liver cell, a brain cell, a gall bladder cell, a heart cell, or a kidney cell, but preferably a liver cell. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell, including human cell in a chimeric non-human animal, or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), or a non-primate cell. In certain embodiments, the cell is a human cell, e.g., a human liver cell. In the methods of the invention, PNPLA3 expression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the level of detection of the assay.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PNPLA3 gene of the mammal to which the RNAi agent is to be administered. The composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intramuscular injection.

In one aspect, the present invention also provides methods for inhibiting the expression of an PNPLA3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a PNPLA3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art and by methods, e.g. qRT-PCR, described herein, e.g., in Example 2. Reduction in protein production can be assessed by any methods known it the art, e.g. ELISA. In certain embodiments, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the PNPLA3 gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the PNPLA3 protein expression.

The present invention further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with a PNPLA3-associated disorder, such as, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

The present invention further provides methods of prophylaxis in a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction of PNPLA3 expression, in a prophylactically effective amount of an iRNA targeting a PNPLA3 gene or a pharmaceutical composition comprising an iRNA targeting a PNPLA3 gene.

In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in PNPLA3 expression, e.g., a PNPLA3-associated disease, such as a chronic fibro-inflammatory liver disease (e.g., cancer, e.g., hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD). In one embodiment, the chronic fibro-inflammatory liver disease is NASH.

In one embodiment, a PNPLA3-associated disease is selected from the group consisting of fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

As used herein, “nonalcoholic fatty liver disease,” used interchangeably with the term “NAFLD,” refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol ingestion per day. NAFLD is the most common liver disease in the United States, and is commonly associated with insulin resistance/type 2 diabetes mellitus and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which are incorporated herein by reference.

As used herein, the terms “steatosis,” “hepatic steatosis,” and “fatty liver disease” refer to the accumulation of triglycerides and other fats in the liver cells.

As used herein, the term “Nonalcoholic steatohepatitis” or “NASH” refers to liver inflammation and damage caused by a buildup of fat in the liver. NASH is part of a group of conditions called nonalcoholic fatty liver disease (NAFLD). NASH resembles alcoholic liver disease, but occurs in people who drink little or no alcohol. The major feature in NASH is fat in the liver, along with inflammation and damage. Most people with NASH feel well and are not aware that they have a liver problem. Nevertheless, NASH can be severe and can lead to cirrhosis, in which the liver is permanently damaged and scarred and no longer able to work properly. NASH is usually first suspected in a person who is found to have elevations in liver tests that are included in routine blood test panels, such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST). When further evaluation shows no apparent reason for liver disease (such as medications, viral hepatitis, or excessive use of alcohol) and when x rays or imaging studies of the liver show fat, NASH is suspected. The only means of proving a diagnosis of NASH and separating it from simple fatty liver is a liver biopsy.

As used herein, the term “cirrhosis,” defined histologically, is a diffuse hepatic process characterized by fibrosis and conversion of the normal liver architecture into structurally abnormal nodules.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from an inhibition of PNPLA3 gene expression are subjects susceptible to or diagnosed with an PNPLA3-associated disorder, such as fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

In an embodiment, the method includes administering a composition featured herein such that expression of the target a PNPLA3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 months per dose. In certain embodiments, the composition is administered once every 3-6 months.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target a PNPLA3 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention may result prevention or treatment of a PNPLA3-associated disorder, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The iRNA is preferably administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired dose of iRNA to a subject. The injections may be repeated over a period of time.

The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as once per month to once a year. In certain embodiments, the iRNA is administered about once per month to about once every three months, or about once every three months to about once every six months.

The invention further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of PNPLA3 gene expression, e.g., a subject having an PNPLA3-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

Accordingly, in some aspects of the invention, the methods which include either a single iRNA agent of the invention, further include administering to the subject one or more additional therapeutic agents.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

Examples of additional therapeutic agents include those known to treat hypertriglyceridemia and other diseases that are caused by, associated with or are a consequence of hypertriglyceridemia. For example, an iRNA featured in the invention can be administered with, e.g., Examples of such agents include, but are not limited to an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., 5-8921, from Shionogi), a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or fish oil. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™) colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PNPLA3 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (AC AT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioMsrieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst).

Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapies include, e.g., AdGWEGF 121.10 (GenVec), ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein IIb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PNPLA3 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypertriglyceridemia, and suitable for administration in combination with a dsRNA targeting PNPLA3 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).

In some embodiments, an iRNA featured in the invention can be administered with, e.g., pyridoxine, an ACE inhibitor (angiotensin converting enzyme inhibitors), e.g., benazepril (Lotensin); an angiotensin II receptor antagonist (ARB) (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), e.g., Candesartan (Atacand); an HMG-CoA reductase inhibitor (e.g., a statin); calcium binding agents, e.g., Sodium cellulose phosphate (Calcibind); diuretics, e.g., thiazide diuretics, such as hydrochlorothiazide (Microzide); an insulin sensitizer, such as the PPARγ agonist pioglitazone, a glp-1r agonist, such as liraglutatide, vitamin E, an SGLT2 inhibitor, a DPPIV inhibitor, and kidney/liver transplant; or a combination of any of the foregoing.

In one embodiment, an iRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)). In one embodiment, the iRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered at the same time.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

In one embodiment, an iRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)). In one embodiment, the iRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered at the same time.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

VIII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof).

Such kits include one or more dsRNA agent(s) and instructions for use, e.g., instructions for administering a prophylactically or therapeutically effective amount of a dsRNA agent(s). The dsRNA agent may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the dsRNA agent (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of PNPLA3 (e.g., means for measuring the inhibition of PNPLA3 mRNA, PNPLA3 protein, and/or PNPLA3 activity). Such means for measuring the inhibition of PNPLA3 may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the invention may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.

In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container, e.g., a vial or a pre-filled syringe. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal Sequence Listing and Figures, are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Design

siRNAs targeting the human Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene (human: NCBI refseqID NM_025225.2; NCBI GeneID: 80339) were designed using custom R and Python scripts. The human NM_025225.2 REFSEQ mRNA, has a length of 2805 bases.

Detailed lists of the unmodified PNPLA3 sense and antisense strand nucleotide sequences are shown in Tables 2, 4, 6, 8, and 10. Detailed lists of the modified PNPLA3 sense and antisense strand nucleotide sequences are shown in Tables 3, 5, 7, 9, 11, 21, 24, 27, and 30.

It is to be understood that, throughout the application, a duplex name without a decimal is equivalent to a duplex name with a decimal which merely references the batch number of the duplex. For example, AD-959917 is equivalent to AD-959917.1.

siRNA Synthesis

siRNAs were designed, synthesized, and prepared using methods known in the art.

Briefly, siRNA sequences were synthesized on a 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. The solid support was controlled pore glass (500-1000 Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universal solid support (AM Chemicals), or the first nucleotide of interest. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, Wis.), Hongene (China), or Chemgenes (Wilmington, Mass., USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs.

Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”).

Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 μL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2′-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 μL of dimethyl sulfoxide (DMSO) and 300 μL TEA.3HF and the solution was incubated for approximately 30 mins at 60° C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively.

Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 μM in 1× PBS in 96 well plates, the plate sealed, incubated at 100° C. for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.

Example 2. In Vitro Screening Methods

Hep3B Cell Culture and 384-Well Transfections

Hep3b cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of each siRNA duplex to an individual well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Eighty μl of complete growth media without antibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done using 8×5-fold serial dilutions over the range of 10 nM to 128 μM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen™, Part #. 610-12)

Cells were lysed in 75 μl of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 90 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 10 μL RT mixture was added to each well, as described below.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.61 of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

Real Time PCR

Two microlitre (μl) of cDNA were added to a master mix containing 0.5 μl of human GAPDH TaqMan Probe (4326317E), 0.5 μl human PNPLA3, 2 μl nuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche).

To calculate relative fold change, data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or mock-transfected. The sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 18) and antisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 19).

In Vitro Dual-Luciferase and Endogenous Screening Assays

Cos-7 cells (ATCC, Manassas, Va.) are grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Single-dose experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM. siRNA and psiCHECK2-PNPLA3 (GenBank Accession No. NM_025225.2) plasmid transfections were carried out with a plasmid containing the 3′ untranslated region (UTR). Transfection was carried out by adding 5 μL of siRNA duplexes and 5 μL (5 ng) of psiCHECK2 plasmid per well along with 4.9 μL of Opti-MEM plus 0.1 μL of Lipofectamine 2000 per well (Invitrogen, Carlsbad Calif. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the cells which are re-suspended in 35 μL of fresh complete media. The transfected cells were incubated at 37° C. in an atmosphere of 5% CO₂.

Forty-eight hours after the siRNAs and psiCHECK2 plasmid are transfected; Firefly (transfection control) and Renilla (fused to PNPLA3 target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μL of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture was incubated at room temperature for 30 minutes before luminescence (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 μL of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (PNPLA3) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done with n=4.

Cell Culture and Transfections

Cells were transfected by adding 4.9 μL of Opti-MEM plus 0.1 μL of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μL of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Forty μL of MEDIA containing ˜5×10³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM. Transfection experiments were performed in Cos 7 cells.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μL of Lysis/Binding Buffer and 10 μL of lysis buffer containing 3 μL of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μL Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μL Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μL of a master mix containing 1 μL 10× Buffer, 0.4 μL 25× dNTPs, 1 μL 10× Random primers, 0.5 μL Reverse Transcriptase, 0.5 μL RNase inhibitor and 6.6 μL of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hour incubation at 37° C.

Real Time PCR

Two μL of cDNA were added to a master mix containing 0.5 μL of human or mouse GAPDH TaqMan Probe (ThermoFisher cat 4352934E or 4351309) and 0.5 μL of appropriate PNPLA3 probe (commercially available, e.g., from Thermo Fisher) and 5 μL Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested with N=4 and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

The results of the screening of the dsRNA agents listed in Tables 2 and 3 in Cos 7 cells are shown in Table 12. The results of the screening of the dsRNA agents listed in Tables 4 and 5 in Cos 7 cells are shown in Table 13. The results of the screening of the dsRNA agents listed in Tables 4 and 5 in Hep3B cells are shown in Table 14. The results of the screening of the dsRNA agents listed in Tables 6 and 7 in Cos 7 cells are shown in Table 15. The results of the screening of the dsRNA agents listed in Tables 8 and 9 in Cos 7 cells are shown in Table 16. The results of the screening of the dsRNA agents listed in Tables 8 and 9 in Hep3B cells are shown in Table 17. The results of the screening of the dsRNA agents listed in Tables 10 and 11 in Cos 7 cells are shown in Table 18. The results of the screening of the dsRNA agents listed in Tables 10 and 11 in Hep3B cells are shown in Table 19.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide, modified or unmodified a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L10 N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol) L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3) Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose) Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydro- furane-5-phosphate) (Agn) Adenosine-glycol nucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn) Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphonate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine-3′-phosphate dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine dUs 2′-deoxyuridine-3′-phosphorothioate (C2p) cytidine-2′-phosphate (G2p) guanosine-2′-phosphate (U2p) uridine-2′-phosphate (A2p) adenosine-2′-phosphate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate

TABLE 2 Unmodified Sense and Antisense Strand Sequences  of PNPLA3 dsRNA Agents SEQ  ID  Range in Duplex Name Sense Sequence 5' to 3' NO: NM_025225.2 AD-517197.1 CAUGAGCAAGAUUUGCAACUU 20 1187-1207 AD-516851.1 UGAUGCCAAAACAACCAUCAU 21 701-721 AD-516748.1 AAAGACGAAGUCGUGGAUGCU 22 576-596 AD-517234.1 GGUGGAUACAUGAGCAAGAUU 23 1179-1199 AD-517354.1 UCCAGAUAUGCCCGACGAUGU 24 1301-1321 AD-517257.1 AACUUGCUACCCAUUAGGAUU 25 1203-1223 AD-516739.1 UCGGUCCAAAGACGAAGUCGU 26 569-589 AD-517258.1 ACUUGCUACCCAUUAGGAUAU 27 1204-1224 AD-516629.1 CUUAAGCAAGUUCCUCCGACU 28 440-460 AD-516972.1 CUGGGAGAGAUAUGCCUUCGU 29 873-893 AD-517623.1 UCCCAUCUUUGUGCAGCUACU 30 1669-1689 AD-516733.1 CUGACUUUCGGUCCAAAGACU 31 562-582 AD-517985.1 ACACCUUUUUCACCUAACUAU 32 2178-2198 AD-516827.1 GUGAGUGACAACGUACCCUUU 33 678-698 AD-516917.1 CAAGCUCAGUCUACGCCUCUU 34 797-817 AD-516973.1 UGGGAGAGAUAUGCCUUCGAU 35 874-894 AD-516978.1 GAGAUAUGCCUUCGAGGAUAU 36 879-899 AD-517310.1 GUGGAAUCUGCCAUUGCGAUU 37 1257-1277 AD-516828.1 UGAGUGACAACGUACCCUUCU 38 679-699 AD-517249.1 CUUGCUACCCAUUAGGAUAAU 39 1205-1225 AD-517196.1 GUGGAUACAUGAGCAAGAUUU 40 1180-1200 AD-517322.1 AUUGCGAUUGUCCAGAGACUU 41 1269-1289 AD-517319.1 GCCAUUGCGAUUGUCCAGAGU 42 1266-1286 AD-516822.1 GAGGAGUGAGUGACAACGUAU 43 673-693 AD-516826.1 AGUGAGUGACAACGUACCCUU 44 677-697 AD-516824.1 GGAGUGAGUGACAACGUACCU 45 675-695 AD-517517.1 UUGGGCAAUAAAGUACCUGCU 46 1545-1565 AD-517758.1 AUGCGUUAAUUCAGCUGGUUU 47 1824-1844 AD-516940.1 CAGGGAACCUCUACCUUCUCU 48 820-840 AD-517318.1 UGCCAUUGCGAUUGUCCAGAU 49 1265-1285 AD-517321.1 CAUUGCGAUUGUCCAGAGACU 50 1268-1288 AD-516747.1 CAAAGACGAAGUCGUGGAUGU 51 575-595 AD-516737.1 CUUUCGGUCCAAAGACGAAGU 52 566-586 AD-516742.1 CGGUCCAAAGACGAAGUCGUU 53 570-590 AD-516977.1 AGAGAUAUGCCUUCGAGGAUU 54 878-898 AD-516823.1 AGGAGUGAGUGACAACGUACU 55 674-694 AD-516871.1 AUCUGCCCUAAAGUCAAGUCU 56 750-770 AD-516771.1 CUUCUACAGUGGCCUUAUCCU 57 620-640 AD-517757.1 CAUGCGUUAAUUCAGCUGGUU 58 1823-1843 AD-516745.1 UCCAAAGACGAAGUCGUGGAU 59 573-593 AD-517830.1 GUGGCCCUAUUAAUGGUCAGU 60 1895-1915 AD-516970.1 UGCUGGGAGAGAUAUGCCUUU 61 871-891 AD-517768.1 UCAGCUGGUUGGGAAAUGACU 62 1834-1854 AD-517259.1 UUGCUACCCAUUAGGAUAAUU 63 1206-1226 AD-516979.1 AGAUAUGCCUUCGAGGAUAUU 64 880-900 AD-516971.1 GCUGGGAGAGAUAUGCCUUCU 65 872-892 AD-517838.1 UUAAUGGUCAGACUGUUCCAU 66 1904-1924 AD-516743.1 GGUCCAAAGACGAAGUCGUGU 67 571-591 AD-516980.1 GAUAUGCCUUCGAGGAUAUUU 68 881-901 AD-517771.1 GCUGGUUGGGAAAUGACACCU 69 1837-1857 AD-516772.1 UUCUACAGUGGCCUUAUCCCU 70 621-641 AD-517836.1 UAUUAAUGGUCAGACUGUUCU 71 1902-1922 AD-516741.1 UUCGGUCCAAAGACGAAGUCU 72 568-588 AD-517353.1 UUCCAGAUAUGCCCGACGAUU 73 1300-1320 AD-517979.1 UUUAGAACACCUUUUUCACCU 74 2172-2192 AD-516937.1 GCACAGGGAACCUCUACCUUU 75 817-837 AD-516976.1 GAGAGAUAUGCCUUCGAGGAU 76 877-897 AD-516872.1 UCUGCCCUAAAGUCAAGUCCU 77 751-771 AD-517256.1 CAACUUGCUACCCAUUAGGAU 78 1202-1222 AD-516825.1 GAGUGAGUGACAACGUACCCU 79 676-696 AD-516735.1 GACUUUCGGUCCAAAGACGAU 80 564-584 AD-516588.1 GGCCAGGAGUCGGAACAUUGU 81 398-418 AD-516738.1 UUUCGGUCCAAAGACGAAGUU 82 567-587 AD-517314.1 AAUCUGCCAUUGCGAUUGUCU 83 1261-1281 AD-517805.1 CAGAGGGUCCCUUACUGACUU 84 1870-1890 AD-517685.1 UUGGUUUUAUGAAAAGCUAGU 85 1751-1771 AD-517831.1 UGGCCCUAUUAAUGGUCAGAU 86 1896-1916 AD-516830.1 AGUGACAACGUACCCUUCAUU 87 681-701 AD-517837.1 AUUAAUGGUCAGACUGUUCCU 88 1903-1923 AD-517633.1 GUGCAGCUACCUCCGCAUUGU 89 1679-1699 AD-516855.1 GCCAAAACAACCAUCACCGUU 90 705-725 AD-516688.1 AACGUUCUGGUGUCUGACUUU 91 549-569 AD-516630.1 UUAAGCAAGUUCCUCCGACAU 92 441-461 AD-516835.1 CAACGUACCCUUCAUUGAUGU 93 686-706 AD-516832.1 UGACAACGUACCCUUCAUUGU 94 683-703 AD-517834.1 CCCUAUUAAUGGUCAGACUGU 95 1899-1919 AD-516734.1 UGACUUUCGGUCCAAAGACGU 96 563-583 AD-517228.1 GACAAAGGUGGAUACAUGAGU 97 1173-1193 AD-516736.1 ACUUUCGGUCCAAAGACGAAU 98 565-585 AD-517646.1 CGCAUUGCUGUGUAGUGACCU 99 1692-1712 AD-517744.1 UCUAAUACAUCAGCAUGCGUU 100 1810-1830 AD-517509.1 ACUUCUUCUUGGGCAAUAAAU 101 1537-1557 AD-517746.1 UAAUACAUCAGCAUGCGUUAU 102 1812-1832 AD-516752.1 ACGAAGUCGUGGAUGCCUUGU 103 580-600 AD-516746.1 CCAAAGACGAAGUCGUGGAUU 104 574-594 AD-517227.1 AGACAAAGGUGGAUACAUGAU 105 1172-1192 AD-516751.1 GACGAAGUCGUGGAUGCCUUU 106 579-599 AD-517042.1 CAUCCUCAGAAGGGAUGGAUU 107 964-984 AD-517571.1 UGAGUCACUUGAGGAGGCGAU 108 1617-1637 AD-517197.1 AAGUUGCAAAUCUUGCUCAUGUA 109 1185-1207 AD-516851.1 AUGAUGGUUGUUUUGGCAUCAAU 110 699-721 AD-516748.1 AGCAUCCACGACUUCGUCUUUGG 111 574-596 AD-517234.1 AAUCUUGCUCAUGUAUCCACCUU 112 1177-1199 AD-517354.1 ACAUCGTCGGGCAUAUCUGGAAG 113 1299-1321 AD-517257.1 AAUCCUAAUGGGUAGCAAGUUGC 114 1201-1223 AD-516739.1 ACGACUTCGUCUUUGGACCGAAA 115 567-589 AD-517258.1 AUAUCCTAAUGGGUAGCAAGUUG 116 1202-1224 AD-516629.1 AGUCGGAGGAACUUGCUUAAGUU 117 438-460 AD-516972.1 ACGAAGGCAUAUCUCUCCCAGCA 118 871-893 AD-517623.1 AGUAGCTGCACAAAGAUGGGAAA 119 1667-1689 AD-516733.1 AGUCUUTGGACCGAAAGUCAGAC 120 560-582 AD-517985.1 AUAGUUAGGUGAAAAAGGUGUUC 121 2176-2198 AD-516827.1 AAAGGGTACGUUGUCACUCACUC 122 676-698 AD-516917.1 AAGAGGCGUAGACUGAGCUUGGU 123 795-817 AD-516973.1 AUCGAAGGCAUAUCUCUCCCAGC 124 872-894 AD-516978.1 AUAUCCTCGAAGGCAUAUCUCUC 125 877-899 AD-517310.1 AAUCGCAAUGGCAGAUUCCACAG 126 1255-1277 AD-516828.1 AGAAGGGUACGUUGUCACUCACU 127 677-699 AD-517249.1 AUUAUCCUAAUGGGUAGCAAGUU 128 1203-1225 AD-517196.1 AAAUCUTGCUCAUGUAUCCACCU 129 1178-1200 AD-517322.1 AAGUCUCUGGACAAUCGCAAUGG 130 1267-1289 AD-517319.1 ACUCUGGACAAUCGCAAUGGCAG 131 1264-1286 AD-516822.1 AUACGUTGUCACUCACUCCUCCA 132 671-693 AD-516826.1 AAGGGUACGUUGUCACUCACUCC 133 675-697 AD-516824.1 AGGUACGUUGUCACUCACUCCUC 134 673-695 AD-517517.1 AGCAGGTACUUUAUUGCCCAAGA 135 1543-1565 AD-517758.1 AAACCAGCUGAAUUAACGCAUGC 136 1822-1844 AD-516940.1 AGAGAAGGUAGAGGUUCCCUGUG 137 818-840 AD-517318.1 AUCUGGACAAUCGCAAUGGCAGA 138 1263-1285 AD-517321.1 AGUCUCTGGACAAUCGCAAUGGC 139 1266-1288 AD-516747.1 ACAUCCACGACUUCGUCUUUGGA 140 573-595 AD-516737.1 ACUUCGTCUUUGGACCGAAAGUC 141 564-586 AD-516742.1 AACGACTUCGUCUUUGGACCGAA 142 568-590 AD-516977.1 AAUCCUCGAAGGCAUAUCUCUCC 143 876-898 AD-516823.1 AGUACGTUGUCACUCACUCCUCC 144 672-694 AD-516871.1 AGACUUGACUUUAGGGCAGAUGU 145 748-770 AD-516771.1 AGGAUAAGGCCACUGUAGAAGGG 146 618-640 AD-517757.1 AACCAGCUGAAUUAACGCAUGCU 147 1821-1843 AD-516745.1 AUCCACGACUUCGUCUUUGGACC 148 571-593 AD-517830.1 ACUGACCAUUAAUAGGGCCACGA 149 1893-1915 AD-516970.1 AAAGGCAUAUCUCUCCCAGCACC 150 869-891 AD-517768.1 AGUCAUTUCCCAACCAGCUGAAU 151 1832-1854 AD-517259.1 AAUUAUCCUAAUGGGUAGCAAGU 152 1204-1226 AD-516979.1 AAUAUCCUCGAAGGCAUAUCUCU 153 878-900 AD-516971.1 AGAAGGCAUAUCUCUCCCAGCAC 154 870-892 AD-517838.1 AUGGAACAGUCUGACCAUUAAUA 155 1902-1924 AD-516743.1 ACACGACUUCGUCUUUGGACCGA 156 569-591 AD-516980.1 AAAUAUCCUCGAAGGCAUAUCUC 157 879-901 AD-517771.1 AGGUGUCAUUUCCCAACCAGCUG 158 1835-1857 AD-516772.1 AGGGAUAAGGCCACUGUAGAAGG 159 619-641 AD-517836.1 AGAACAGUCUGACCAUUAAUAGG 160 1900-1922 AD-516741.1 AGACUUCGUCUUUGGACCGAAAG 161 566-588 AD-517353.1 AAUCGUCGGGCAUAUCUGGAAGC 162 1298-1320 AD-517979.1 AGGUGAAAAAGGUGUUCUAAAAU 163 2170-2192 AD-516937.1 AAAGGUAGAGGUUCCCUGUGCAG 164 815-837 AD-516976.1 AUCCUCGAAGGCAUAUCUCUCCC 165 875-897 AD-516872.1 AGGACUTGACUUUAGGGCAGAUG 166 749-771 AD-517256.1 AUCCUAAUGGGUAGCAAGUUGCA 167 1200-1222 AD-516825.1 AGGGUACGUUGUCACUCACUCCU 168 674-696 AD-516735.1 AUCGUCTUUGGACCGAAAGUCAG 169 562-584 AD-516588.1 ACAAUGTUCCGACUCCUGGCCUU 170 396-418 AD-516738.1 AACUUCGUCUUUGGACCGAAAGU 171 565-587 AD-517314.1 AGACAATCGCAAUGGCAGAUUCC 172 1259-1281 AD-517805.1 AAGUCAGUAAGGGACCCUCUGCA 173 1868-1890 AD-517685.1 ACUAGCTUUUCAUAAAACCAACU 174 1749-1771 AD-517831.1 AUCUGACCAUUAAUAGGGCCACG 175 1894-1916 AD-516830.1 AAUGAAGGGUACGUUGUCACUCA 176 679-701 AD-517837.1 AGGAACAGUCUGACCAUUAAUAG 177 1901-1923 AD-517633.1 ACAAUGCGGAGGUAGCUGCACAA 178 1677-1699 AD-516855.1 AACGGUGAUGGUUGUUUUGGCAU 179 703-725 AD-516688.1 AAAGUCAGACACCAGAACGUUUU 180 547-569 AD-516630.1 AUGUCGGAGGAACUUGCUUAAGU 181 439-461 AD-516835.1 ACAUCAAUGAAGGGUACGUUGUC 182 684-706 AD-516832.1 ACAAUGAAGGGUACGUUGUCACU 183 681-703 AD-517834.1 ACAGUCTGACCAUUAAUAGGGCC 184 1897-1919 AD-516734.1 ACGUCUTUGGACCGAAAGUCAGA 185 561-583 AD-517228.1 ACUCAUGUAUCCACCUUUGUCUU 186 1171-1193 AD-516736.1 AUUCGUCUUUGGACCGAAAGUCA 187 563-585 AD-517646.1 AGGUCACUACACAGCAAUGCGGA 188 1690-1712 AD-517744.1 AACGCATGCUGAUGUAUUAGAGU 189 1808-1830 AD-517509.1 AUUUAUTGCCCAAGAAGAAGUUC 190 1535-1557 AD-517746.1 AUAACGCAUGCUGAUGUAUUAGA 191 1810-1832 AD-516752.1 ACAAGGCAUCCACGACUUCGUCU 192 578-600 AD-516746.1 AAUCCACGACUUCGUCUUUGGAC 193 572-594 AD-517227.1 AUCAUGTAUCCACCUUUGUCUUU 194 1170-1192 AD-516751.1 AAAGGCAUCCACGACUUCGUCUU 195 577-599 AD-517042.1 AAUCCATCCCUUCUGAGGAUGAC 196 962-984 AD-517571.1 AUCGCCTCCUCAAGUGACUCACA 197 1615-1637

TABLE 3 Modified Sense and Antisense Strand Sequences  of PNPLA3 dsRNA Agents SEQ ID Duplex Name Sense Sequence 5' to 3' NO: AD-517197.1 csasugagCfaAfGfAfuuugcaacuuL96 198 AD-516851.1 usgsaugcCfaAfAfAfcaaccaucauL96 199 AD-516748.1 asasagacGfaAfGfUfcguggaugcuL96 200 AD-517234.1 gsgsuggaUfaCfAfUfgagcaagauuL96 201 AD-517354.1 uscscagaUfaUfGfCfccgacgauguL96 202 AD-517257.1 asascuugCfuAfCfCfcauuaggauuL96 203 AD-516739.1 uscsggucCfaAfAfGfacgaagucguL96 204 AD-517258.1 ascsuugcUfaCfCfCfauuaggauauL96 205 AD-516629.1 csusuaagCfaAfGfUfuccuccgacuL96 206 AD-516972.1 csusgggaGfaGfAfUfaugccuucguL96 207 AD-517623.1 uscsccauCfuUfUfGfugcagcuacuL96 208 AD-516733.1 csusgacuUfuCfGfGfuccaaagacuL96 209 AD-517985.1 ascsaccuUfuUfUfCfaccuaacuauL96 210 AD-516827.1 gsusgaguGfaCfAfAfcguacccuuuL96 211 AD-516917.1 csasagcuCfaGfUfCfuacgccucuuL96 212 AD-516973.1 usgsggagAfgAfUfAfugccuucgauL96 213 AD-516978.1 gsasgauaUfgCfCfUfucgaggauauL96 214 AD-517310.1 gsusggaaUfcUfGfCfcauugcgauuL96 215 AD-516828.1 usgsagugAfcAfAfCfguacccuucuL96 216 AD-517249.1 csusugcuAfcCfCfAfuuaggauaauL96 217 AD-517196.1 gsusggauAfcAfUfGfagcaagauuuL96 218 AD-517322.1 asusugcgAfuUfGfUfccagagacuuL96 219 AD-517319.1 gscscauuGfcGfAfUfuguccagaguL96 220 AD-516822.1 gsasggagUfgAfGfUfgacaacguauL96 221 AD-516826.1 asgsugagUfgAfCfAfacguacccuuL96 222 AD-516824.1 gsgsagugAfgUfGfAfcaacguaccuL96 223 AD-517517.1 ususgggcAfaUfAfAfaguaccugcuL96 224 AD-517758.1 asusgcguUfaAfUfUfcagcugguuuL96 225 AD-516940.1 csasgggaAfcCfUfCfuaccuucucuL96 226 AD-517318.1 usgsccauUfgCfGfAfuuguccagauL96 227 AD-517321.1 csasuugcGfaUfUfGfuccagagacuL96 228 AD-516747.1 csasaagaCfgAfAfGfucguggauguL96 229 AD-516737.1 csusuucgGfuCfCfAfaagacgaaguL96 230 AD-516742.1 csgsguccAfaAfGfAfcgaagucguuL96 231 AD-516977.1 asgsagauAfuGfCfCfuucgaggauuL96 232 AD-516823.1 asgsgaguGfaGfUfGfacaacguacuL96 233 AD-516871.1 asuscugcCfcUfAfAfagucaagucuL96 234 AD-516771.1 csusucuaCfaGfUfGfgccuuauccuL96 235 AD-517757.1 csasugcgUfuAfAfUfucagcugguuL96 236 AD-516745.1 uscscaaaGfaCfGfAfagucguggauL96 237 AD-517830.1 gsusggccCfuAfUfUfaauggucaguL96 238 AD-516970.1 usgscuggGfaGfAfGfauaugccuuuL96 239 AD-517768.1 uscsagcuGfgUfUfGfggaaaugacuL96 240 AD-517259.1 ususgcuaCfcCfAfUfuaggauaauuL96 241 AD-516979.1 asgsauauGfcCfUfUfcgaggauauuL96 242 AD-516971.1 gscsugggAfgAfGfAfuaugccuucuL96 243 AD-517838.1 ususaaugGfuCfAfGfacuguuccauL96 244 AD-516743.1 gsgsuccaAfaGfAfCfgaagucguguL96 245 AD-516980.1 gsasuaugCfcUfUfCfgaggauauuuL96 246 AD-517771.1 gscsugguUfgGfGfAfaaugacaccuL96 247 AD-516772.1 ususcuacAfgUfGfGfccuuaucccuL96 248 AD-517836.1 usasuuaaUfgGfUfCfagacuguucuL96 249 AD-516741.1 ususcgguCfcAfAfAfgacgaagucuL96 250 AD-517353.1 ususccagAfuAfUfGfcccgacgauuL96 251 AD-517979.1 ususuagaAfcAfCfCfuuuuucaccuL96 252 AD-516937.1 gscsacagGfgAfAfCfcucuaccuuuL96 253 AD-516976.1 gsasgagaUfaUfGfCfcuucgaggauL96 254 AD-516872.1 uscsugccCfuAfAfAfgucaaguccuL96 255 AD-517256.1 csasacuuGfcUfAfCfccauuaggauL96 256 AD-516825.1 gsasgugaGfuGfAfCfaacguacccuL96 257 AD-516735.1 gsascuuuCfgGfUfCfcaaagacgauL96 258 AD-516588.1 gsgsccagGfaGfUfCfggaacauuguL96 259 AD-516738.1 ususucggUfcCfAfAfagacgaaguuL96 260 AD-517314.1 asasucugCfcAfUfUfgcgauugucuL96 261 AD-517805.1 csasgaggGfuCfCfCfuuacugacuuL96 262 AD-517685.1 ususgguuUfuAfUfGfaaaagcuaguL96 263 AD-517831.1 usgsgcccUfaUfUfAfauggucagauL96 264 AD-516830.1 asgsugacAfaCfGfUfacccuucauuL96 265 AD-517837.1 asusuaauGfgUfCfAfgacuguuccuL96 266 AD-517633.1 gsusgcagCfuAfCfCfuccgcauuguL96 267 AD-516855.1 gscscaaaAfcAfAfCfcaucaccguuL96 268 AD-516688.1 asascguuCfuGfGfUfgucugacuuuL96 269 AD-516630.1 ususaagcAfaGfUfUfccuccgacauL96 270 AD-516835.1 csasacguAfcCfCfUfucauugauguL96 271 AD-516832.1 usgsacaaCfgUfAfCfccuucauuguL96 272 AD-517834.1 cscscuauUfaAfUfGfgucagacuguL96 273 AD-516734.1 usgsacuuUfcGfGfUfccaaagacguL96 274 AD-517228.1 gsascaaaGfgUfGfGfauacaugaguL96 275 AD-516736.1 ascsuuucGfgUfCfCfaaagacgaauL96 276 AD-517646.1 csgscauuGfcUfGfUfguagugaccuL96 277 AD-517744.1 uscsuaauAfcAfUfCfagcaugcguuL96 278 AD-517509.1 ascsuucuUfcUfUfGfggcaauaaauL96 279 AD-517746.1 usasauacAfuCfAfGfcaugcguuauL96 280 AD-516752.1 ascsgaagUfcGfUfGfgaugccuuguL96 281 AD-516746.1 cscsaaagAfcGfAfAfgucguggauuL96 282 AD-517227.1 asgsacaaAfgGfUfGfgauacaugauL96 283 AD-516751.1 gsascgaaGfuCfGfUfggaugccuuuL96 284 AD-517042.1 csasuccuCfaGfAfAfgggauggauuL96 285 AD-517571.1 usgsagucAfcUfUfGfaggaggcgauL96 286 AD-517197.1 asAfsguug(Cgn)aaaucuUfgCfucaugsusa 287 AD-516851.1 asUfsgaug(Ggn)uuguuuUfgGfcaucasasu 288 AD-516748.1 asGfscauc(Cgn)acgacuUfcGfucuuusgsg 289 AD-517234.1 asAfsucuu(Ggn)cucaugUfaUfccaccsusu 290 AD-517354.1 asCfsaucg(Tgn)cgggcaUfaUfcuggasasg 291 AD-517257.1 asAfsuccu(Agn)auggguAfgCfaaguusgsc 292 AD-516739.1 asCfsgacu(Tgn)cgucuuUfgGfaccgasasa 293 AD-517258.1 asUfsaucc(Tgn)aaugggUfaGfcaagususg 294 AD-516629.1 asGfsucgg(Agn)ggaacuUfgCfuuaagsusu 295 AD-516972.1 asCfsgaag(Ggn)cauaucUfcUfcccagscsa 296 AD-517623.1 asGfsuagc(Tgn)gcacaaAfgAfugggasasa 297 AD-516733.1 asGfsucuu(Tgn)ggaccgAfaAfgucagsasc 298 AD-517985.1 asUfsaguu(Agn)ggugaaAfaAfggugususc 299 AD-516827.1 asAfsaggg(Tgn)acguugUfcAfcucacsusc 300 AD-516917.1 asAfsgagg(Cgn)guagacUfgAfgcuugsgsu 301 AD-516973.1 asUfscgaa(Ggn)gcauauCfuCfucccasgsc 302 AD-516978.1 asUfsaucc(Tgn)cgaaggCfaUfaucucsusc 303 AD-517310.1 asAfsucgc(Agn)auggcaGfaUfuccacsasg 304 AD-516828.1 asGfsaagg(Ggn)uacguuGfuCfacucascsu 305 AD-517249.1 asUfsuauc(Cgn)uaauggGfuAfgcaagsusu 306 AD-517196.1 asAfsaucu(Tgn)gcucauGfuAfuccacscsu 307 AD-517322.1 asAfsgucu(Cgn)uggacaAfuCfgcaausgsg 308 AD-517319.1 asCfsucug(Ggn)acaaucGfcAfauggcsasg 309 AD-516822.1 asUfsacgu(Tgn)gucacuCfaCfuccucscsa 310 AD-516826.1 asAfsgggu(Agn)cguuguCfaCfucacuscsc 311 AD-516824.1 asGfsguac(Ggn)uugucaCfuCfacuccsusc 312 AD-517517.1 asGfscagg(Tgn)acuuuaUfuGfcccaasgsa 313 AD-517758.1 asAfsacca(Ggn)cugaauUfaAfcgcausgsc 314 AD-516940.1 asGfsagaa(Ggn)guagagGfuUfcccugsusg 315 AD-517318.1 asUfscugg(Agn)caaucgCfaAfuggcasgsa 316 AD-517321.1 asGfsucuc(Tgn)ggacaaUfcGfcaaugsgsc 317 AD-516747.1 asCfsaucc(Agn)cgacuuCfgUfcuuugsgsa 318 AD-516737.1 asCfsuucg(Tgn)cuuuggAfcCfgaaagsusc 319 AD-516742.1 asAfscgac(Tgn)ucgucuUfuGfgaccgsasa 320 AD-516977.1 asAfsuccu(Cgn)gaaggcAfuAfucucuscsc 321 AD-516823.1 asGfsuacg(Tgn)ugucacUfcAfcuccuscsc 322 AD-516871.1 asGfsacuu(Ggn)acuuuaGfgGfcagausgsu 323 AD-516771.1 asGfsgaua(Agn)ggccacUfgUfagaagsgsg 324 AD-517757.1 asAfsccag(Cgn)ugaauuAfaCfgcaugscsu 325 AD-516745.1 asUfsccac(Ggn)acuucgUfcUfuuggascsc 326 AD-517830.1 asCfsugac(Cgn)auuaauAfgGfgccacsgsa 327 AD-516970.1 asAfsaggc(Agn)uaucucUfcCfcagcascsc 328 AD-517768.1 asGfsucau(Tgn)ucccaaCfcAfgcugasasu 329 AD-517259.1 asAfsuuau(Cgn)cuaaugGfgUfagcaasgsu 330 AD-516979.1 asAfsuauc(Cgn)ucgaagGfcAfuaucuscsu 331 AD-516971.1 asGfsaagg(Cgn)auaucuCfuCfccagcsasc 332 AD-517838.1 asUfsggaa(Cgn)agucugAfcCfauuaasusa 333 AD-516743.1 asCfsacga(Cgn)uucgucUfuUfggaccsgsa 334 AD-516980.1 asAfsauau(Cgn)cucgaaGfgCfauaucsusc 335 AD-517771.1 asGfsgugu(Cgn)auuuccCfaAfccagcsusg 336 AD-516772.1 asGfsggau(Agn)aggccaCfuGfuagaasgsg 337 AD-517836.1 asGfsaaca(Ggn)ucugacCfaUfuaauasgsg 338 AD-516741.1 asGfsacuu(Cgn)gucuuuGfgAfccgaasasg 339 AD-517353.1 asAfsucgu(Cgn)gggcauAfuCfuggaasgsc 340 AD-517979.1 asGfsguga(Agn)aaagguGfuUfcuaaasasu 341 AD-516937.1 asAfsaggu(Agn)gagguuCfcCfugugcsasg 342 AD-516976.1 asUfsccuc(Ggn)aaggcaUfaUfcucucscsc 343 AD-516872.1 asGfsgacu(Tgn)gacuuuAfgGfgcagasusg 344 AD-517256.1 asUfsccua(Agn)uggguaGfcAfaguugscsa 345 AD-516825.1 asGfsggua(Cgn)guugucAfcUfcacucscsu 346 AD-516735.1 asUfscguc(Tgn)uuggacCfgAfaagucsasg 347 AD-516588.1 asCfsaaug(Tgn)uccgacUfcCfuggccsusu 348 AD-516738.1 asAfscuuc(Ggn)ucuuugGfaCfcgaaasgsu 349 AD-517314.1 asGfsacaa(Tgn)cgcaauGfgCfagauuscsc 350 AD-517805.1 asAfsguca(Ggn)uaagggAfcCfcucugscsa 351 AD-517685.1 asCfsuagc(Tgn)uuucauAfaAfaccaascsu 352 AD-517831.1 asUfscuga(Cgn)cauuaaUfaGfggccascsg 353 AD-516830.1 asAfsugaa(Ggn)gguacgUfuGfucacuscsa 354 AD-517837.1 asGfsgaac(Agn)gucugaCfcAfuuaausasg 355 AD-517633.1 asCfsaaug(Cgn)ggagguAfgCfugcacsasa 356 AD-516855.1 asAfscggu(Ggn)augguuGfuUfuuggcsasu 357 AD-516688.1 asAfsaguc(Agn)gacaccAfgAfacguususu 358 AD-516630.1 asUfsgucg(Ggn)aggaacUfuGfcuuaasgsu 359 AD-516835.1 asCfsauca(Agn)ugaaggGfuAfcguugsusc 360 AD-516832.1 asCfsaaug(Agn)aggguaCfgUfugucascsu 361 AD-517834.1 asCfsaguc(Tgn)gaccauUfaAfuagggscsc 362 AD-516734.1 asCfsgucu(Tgn)uggaccGfaAfagucasgsa 363 AD-517228.1 asCfsucau(Ggn)uauccaCfcUfuugucsusu 364 AD-516736.1 asUfsucgu(Cgn)uuuggaCfcGfaaaguscsa 365 AD-517646.1 asGfsguca(Cgn)uacacaGfcAfaugcgsgsa 366 AD-517744.1 asAfscgca(Tgn)gcugauGfuAfuuagasgsu 367 AD-517509.1 asUfsuuau(Tgn)gcccaaGfaAfgaagususc 368 AD-517746.1 asUfsaacg(Cgn)augcugAfuGfuauuasgsa 369 AD-516752.1 asCfsaagg(Cgn)auccacGfaCfuucguscsu 370 AD-516746.1 asAfsucca(Cgn)gacuucGfuCfuuuggsasc 371 AD-517227.1 asUfscaug(Tgn)auccacCfuUfugucususu 372 AD-516751.1 asAfsaggc(Agn)uccacgAfcUfucgucsusu 373 AD-517042.1 asAfsucca(Tgn)cccuucUfgAfggaugsasc 374 AD-517571.1 asUfscgcc(Tgn)ccucaaGfuGfacucascsa 375 AD-517197.1 UACAUGAGCAAGAUUUGCAACUU 376 AD-516851.1 AUUGAUGCCAAAACAACCAUCAC 377 AD-516748.1 CCAAAGACGAAGUCGUGGAUGCC 378 AD-517234.1 AAGGUGGAUACAUGAGCAAGAUU 379 AD-517354.1 CUUCCAGAUAUGCCCGACGAUGU 380 AD-517257.1 GCAACUUGCUACCCAUUAGGAUA 381 AD-516739.1 UUUCGGUCCAAAGACGAAGUCGU 382 AD-517258.1 CAACUUGCUACCCAUUAGGAUAA 383 AD-516629.1 AACUUAAGCAAGUUCCUCCGACA 384 AD-516972.1 UGCUGGGAGAGAUAUGCCUUCGA 385 AD-517623.1 UUUCCCAUCUUUGUGCAGCUACC 386 AD-516733.1 GUCUGACUUUCGGUCCAAAGACG 387 AD-517985.1 GAACACCUUUUUCACCUAACUAA 388 AD-516827.1 GAGUGAGUGACAACGUACCCUUC 389 AD-516917.1 ACCAAGCUCAGUCUACGCCUCUG 390 AD-516973.1 GCUGGGAGAGAUAUGCCUUCGAG 391 AD-516978.1 GAGAGAUAUGCCUUCGAGGAUAU 392 AD-517310.1 CUGUGGAAUCUGCCAUUGCGAUU 393 AD-516828.1 AGUGAGUGACAACGUACCCUUCA 394 AD-517249.1 AACUUGCUACCCAUUAGGAUAAU 395 AD-517196.1 AGGUGGAUACAUGAGCAAGAUUU 396 AD-517322.1 CCAUUGCGAUUGUCCAGAGACUG 397 AD-517319.1 CUGCCAUUGCGAUUGUCCAGAGA 398 AD-516822.1 UGGAGGAGUGAGUGACAACGUAC 399 AD-516826.1 GGAGUGAGUGACAACGUACCCUU 400 AD-516824.1 GAGGAGUGAGUGACAACGUACCC 401 AD-517517.1 UCUUGGGCAAUAAAGUACCUGCU 402 AD-517758.1 GCAUGCGUUAAUUCAGCUGGUUG 403 AD-516940.1 CACAGGGAACCUCUACCUUCUCU 404 AD-517318.1 UCUGCCAUUGCGAUUGUCCAGAG 405 AD-517321.1 GCCAUUGCGAUUGUCCAGAGACU 406 AD-516747.1 UCCAAAGACGAAGUCGUGGAUGC 407 AD-516737.1 GACUUUCGGUCCAAAGACGAAGU 408 AD-516742.1 UUCGGUCCAAAGACGAAGUCGUG 409 AD-516977.1 GGAGAGAUAUGCCUUCGAGGAUA 410 AD-516823.1 GGAGGAGUGAGUGACAACGUACC 411 AD-516871.1 ACAUCUGCCCUAAAGUCAAGUCC 412 AD-516771.1 CCCUUCUACAGUGGCCUUAUCCC 413 AD-517757.1 AGCAUGCGUUAAUUCAGCUGGUU 414 AD-516745.1 GGUCCAAAGACGAAGUCGUGGAU 415 AD-517830.1 UCGUGGCCCUAUUAAUGGUCAGA 416 AD-516970.1 GGUGCUGGGAGAGAUAUGCCUUC 417 AD-517768.1 AUUCAGCUGGUUGGGAAAUGACA 418 AD-517259.1 ACUUGCUACCCAUUAGGAUAAUG 419 AD-516979.1 AGAGAUAUGCCUUCGAGGAUAUU 420 AD-516971.1 GUGCUGGGAGAGAUAUGCCUUCG 421 AD-517838.1 UAUUAAUGGUCAGACUGUUCCAG 422 AD-516743.1 UCGGUCCAAAGACGAAGUCGUGG 423 AD-516980.1 GAGAUAUGCCUUCGAGGAUAUUU 424 AD-517771.1 CAGCUGGUUGGGAAAUGACACCA 425 AD-516772.1 CCUUCUACAGUGGCCUUAUCCCU 426 AD-517836.1 CCUAUUAAUGGUCAGACUGUUCC 427 AD-516741.1 CUUUCGGUCCAAAGACGAAGUCG 428 AD-517353.1 GCUUCCAGAUAUGCCCGACGAUG 429 AD-517979.1 AUUUUAGAACACCUUUUUCACCU 430 AD-516937.1 CUGCACAGGGAACCUCUACCUUC 431 AD-516976.1 GGGAGAGAUAUGCCUUCGAGGAU 432 AD-516872.1 CAUCUGCCCUAAAGUCAAGUCCA 433 AD-517256.1 UGCAACUUGCUACCCAUUAGGAU 434 AD-516825.1 AGGAGUGAGUGACAACGUACCCU 435 AD-516735.1 CUGACUUUCGGUCCAAAGACGAA 436 AD-516588.1 AAGGCCAGGAGUCGGAACAUUGG 437 AD-516738.1 ACUUUCGGUCCAAAGACGAAGUC 438 AD-517314.1 GGAAUCUGCCAUUGCGAUUGUCC 439 AD-517805.1 UGCAGAGGGUCCCUUACUGACUG 440 AD-517685.1 AGUUGGUUUUAUGAAAAGCUAGG 441 AD-517831.1 CGUGGCCCUAUUAAUGGUCAGAC 442 AD-516830.1 UGAGUGACAACGUACCCUUCAUU 443 AD-517837.1 CUAUUAAUGGUCAGACUGUUCCA 444 AD-517633.1 UUGUGCAGCUACCUCCGCAUUGC 445 AD-516855.1 AUGCCAAAACAACCAUCACCGUG 446 AD-516688.1 AAAACGUUCUGGUGUCUGACUUU 447 AD-516630.1 ACUUAAGCAAGUUCCUCCGACAG 448 AD-516835.1 GACAACGUACCCUUCAUUGAUGC 449 AD-516832.1 AGUGACAACGUACCCUUCAUUGA 450 AD-517834.1 GGCCCUAUUAAUGGUCAGACUGU 451 AD-516734.1 UCUGACUUUCGGUCCAAAGACGA 452 AD-517228.1 AAGACAAAGGUGGAUACAUGAGC 453 AD-516736.1 UGACUUUCGGUCCAAAGACGAAG 454 AD-517646.1 UCCGCAUUGCUGUGUAGUGACCC 455 AD-517744.1 ACUCUAAUACAUCAGCAUGCGUU 456 AD-517509.1 GAACUUCUUCUUGGGCAAUAAAG 457 AD-517746.1 UCUAAUACAUCAGCAUGCGUUAA 458 AD-516752.1 AGACGAAGUCGUGGAUGCCUUGG 459 AD-516746.1 GUCCAAAGACGAAGUCGUGGAUG 460 AD-517227.1 AAAGACAAAGGUGGAUACAUGAG 461 AD-516751.1 AAGACGAAGUCGUGGAUGCCUUG 462 AD-517042.1 GUCAUCCUCAGAAGGGAUGGAUC 463 AD-517571.1 UGUGAGUCACUUGAGGAGGCGAG 464

TABLE 4 Unmodified Sense and Antisense Strand  Sequences of PNPLA3 dsRNA Agents SEQ Duplex Sense Sequence 5'  ID Range in Name to 3' NO: NM_025225.2 AD-67605.6 ACCUGUUGAAUUUUGUAUUAU 465 2245-2265 AD-520101.1 UACCUGUUGAAUUUUGUAUUU 466 2244-2264 AD-520098.1 GUUGUUACCUGUUGAAUUUUU 467 2239-2259 AD-67575.6 UUACCUGUUGAAUUUUGUAUU 468 2243-2263 AD-520467.1 UUGAACCUGGCUUAUUUUCUU 469 2714-2734 AD-520064.1 CUUUUUCACCUAACUAAAAUU 470 2182-2202 AD-520099.1 UGUUACCUGUUGAAUUUUGUU 471 2241-2261 AD-520466.1 CUUGAACCUGGCUUAUUUUCU 472 2713-2733 AD-519351.1 AGGAUAAUGUCUUAUGUAAUU 473 1218-1238 AD-520065.1 UUUUUCACCUAACUAAAAUAU 474 2183-2203 AD-520069.1 CACCUAACUAAAAUAAUGUUU 475 2188-2208 AD-519828.1 CAUCAGCAUGCGUUAAUUCAU 476 1817-1837 AD-520035.1 GGUAACAAGAUGAUAAUCUAU 477 2146-2166 AD-520067.1 UUUCACCUAACUAAAAUAAUU 478 2185-2205 AD-75289.2 UUCACCUAACUAAAAUAAUGU 479 2186-2206 AD-520125.1 GUGAGAUGUUAGUAGAAUAAU 480 2274-2294 AD-520018.1 GAUAACCUUGACUACUAAAAU 481 2110-2130 AD-520062.1 ACCUUUUUCACCUAACUAAAU 482 2180-2200 AD-519754.1 GAGCUGAGUUGGUUUUAUGAU 483 1743-1763 AD-520097.1 CGUUGUUACCUGUUGAAUUUU 484 2238-2258 AD-520352.1 UGAGAUUGCACCAUUUCAUUU 485 2544-2564 AD-519755.1 AGCUGAGUUGGUUUUAUGAAU 486 1744-1764 AD-520063.1 CCUUUUUCACCUAACUAAAAU 487 2181-2201 AD-520066.1 UUUUCACCUAACUAAAAUAAU 488 2184-2204 AD-520068.1 UCACCUAACUAAAAUAAUGUU 489 2187-2207 AD-520465.1 ACUUGAACCUGGCUUAUUUUU 490 2712-2732 AD-519592.1 UCUUCUUGGGCAAUAAAGUAU 491 1540-1560 AD-519591.1 UUCUUCUUGGGCAAUAAAGUU 492 1539-1559 AD-67605.6 AUAAUACAAAAUUCAACAGGUAA 493 2243-2265 AD-520101.1 AAAUACAAAAUUCAACAGGUAAC 494 2242-2264 AD-520098.1 AAAAAUUCAACAGGUAACAACGC 495 2237-2259 AD-67575.6 AAUACAAAAUUCAACAGGUAACA 496 2241-2263 AD-520467.1 AAGAAAAUAAGCCAGGUUCAAGU 497 2712-2734 AD-520064.1 AAUUUUAGUUAGGUGAAAAAGGU 498 2180-2202 AD-520099.1 AACAAAAUUCAACAGGUAACAAC 499 2239-2261 AD-520466.1 AGAAAAUAAGCCAGGUUCAAGUU 500 2711-2733 AD-519351.1 AAUUACAUAAGACAUUAUCCUAA 501 1216-1238 AD-520065.1 AUAUUUUAGUUAGGUGAAAAAGG 502 2181-2203 AD-520069.1 AAACAUUAUUUUAGUUAGGUGAA 503 2186-2208 AD-519828.1 AUGAAUUAACGCAUGCUGAUGUA 504 1815-1837 AD-520035.1 AUAGAUUAUCAUCUUGUUACCCC 505 2144-2166 AD-520067.1 AAUUAUUUUAGUUAGGUGAAAAA 506 2183-2205 AD-75289.2 ACAUUAUUUUAGUUAGGUGAAAA 507 2184-2206 AD-520125.1 AUUAUUCUACUAACAUCUCACUG 508 2272-2294 AD-520018.1 AUUUUAGUAGUCAAGGUUAUCAU 509 2108-2130 AD-520062.1 AUUUAGUUAGGUGAAAAAGGUGU 510 2178-2200 AD-519754.1 AUCAUAAAACCAACUCAGCUCAG 511 1741-1763 AD-520097.1 AAAAUUCAACAGGUAACAACGCU 512 2236-2258 AD-520352.1 AAAUGAAAUGGUGCAAUCUCAGC 513 2542-2564 AD-519755.1 AUUCAUAAAACCAACUCAGCUCA 514 1742-1764 AD-520063.1 AUUUUAGUUAGGUGAAAAAGGUG 515 2179-2201 AD-520066.1 AUUAUUUUAGUUAGGUGAAAAAG 516 2182-2204 AD-520068.1 AACAUUAUUUUAGUUAGGUGAAA 517 2185-2207 AD-520465.1 AAAAAUAAGCCAGGUUCAAGUUG 518 2710-2732 AD-519592.1 AUACUUUAUUGCCCAAGAAGAAG 519 1538-1560 AD-519591.1 AACUUUAUUGCCCAAGAAGAAGU 520 1537-1559

TABLE 5 Modified Sense and Antisense  Strand Sequences of PNPLA3 dsRNA Agents SEQ Duplex ID Name Sense Sequence 5′ to 3′ NO: AD-67605.6 ascscuguUfgAfAfUfuuuguauuauL96 521 AD-520101.1 usasccugUfuGfAfAfuuuuguauuuL96 522 AD-520098.1 gsusuguuAfcCfUfGfuugaauuuuuL96 523 AD-67575.6 ususaccuGfuUfGfAfauuuuguauuL96 524 AD-520467.1 ususgaacCfuGfGfCfuuauuuucuuL96 525 AD-520064.1 csusuuuuCfaCfCfUfaacuaaaauuL96 526 AD-520099.1 usgsuuacCfuGfUfUfgaauuuuguuL96 527 AD-520466.1 csusugaaCfcUfGfGfcuuauuuucuL96 528 AD-519351.1 asgsgauaAfuGfUfCfuuauguaauuL96 529 AD-520065.1 ususuuucAfcCfUfAfacuaaaauauL96 530 AD-520069.1 csasccuaAfcUfAfAfaauaauguuuL96 531 AD-519828.1 csasucagCfaUfGfCfguuaauucauL96 532 AD-520035.1 gsgsuaacAfaGfAfUfgauaaucuauL96 533 AD-520067.1 ususucacCfuAfAfCfuaaaauaauuL96 534 AD-75289.2 ususcaccUfaAfCfUfaaaauaauguL96 535 AD-520125.1 gsusgagaUfgUfUfAfguagaauaauL96 536 AD-520018.1 gsasuaacCfuUfGfAfcuacuaaaauL96 537 AD-520062.1 ascscuuuUfuCfAfCfcuaacuaaauL96 538 AD-519754.1 gsasgcugAfgUfUfGfguuuuaugauL96 539 AD-520097.1 csgsuuguUfaCfCfUfguugaauuuuL96 540 AD-520352.1 usgsagauUfgCfAfCfcauuucauuuL96 541 AD-519755.1 asgscugaGfuUfGfGfuuuuaugaauL96 542 AD-520063.1 cscsuuuuUfcAfCfCfuaacuaaaauL96 543 AD-520066.1 ususuucaCfcUfAfAfcuaaaauaauL96 544 AD-520068.1 uscsaccuAfaCfUfAfaaauaauguuL96 545 AD-520465.1 ascsuugaAfcCfUfGfgcuuauuuuuL96 546 AD-519592.1 uscsuucuUfgGfGfCfaauaaaguauL96 547 AD-519591.1 ususcuucUfuGfGfGfcaauaaaguuL96 548 SEQ Duplex ID Name Antisense Sequence 5′to 3′ NO: AD-67605.6 asUfsaauAfcAfAfaauuCfaAfcaggusasa 549 AD-520101.1 asAfsauaCfaAfAfauucAfaCfagguasasc 550 AD-520098.1 asAfsaaaUfuCfAfacagGfuAfacaacsgsc 551 AD-67575.6 asAfsuacAfaAfAfuucaAfcAfgguaascsa 552 AD-520467.1 asAfsgaaAfaUfAfagccAfgGfuucaasgsu 553 AD-520064.1 asAfsuuuUfaGfUfuaggUfgAfaaaagsgsu 554 AD-520099.1 asAfscaaAfaUfUfcaacAfgGfuaacasasc 555 AD-520466.1 asGfsaaaAfuAfAfgccaGfgUfucaagsusu 556 AD-519351.1 asAfsuuaCfaUfAfagacAfuUfauccusasa 557 AD-520065.1 asUfsauuUfuAfGfuuagGfuGfaaaaasgsg 558 AD-520069.1 asAfsacaUfuAfUfuuuaGfuUfaggugsasa 559 AD-519828.1 asUfsgaaUfuAfAfcgcaUfgCfugaugsusa 560 AD-520035.1 asUfsagaUfuAfUfcaucUfuGfuuaccscsc 561 AD-520067.1 asAfsuuaUfuUfUfaguuAfgGfugaaasasa 562 AD-75289.2 asCfsauuAfuUfUfuaguUfaGfgugaasasa 563 AD-520125.1 asUfsuauUfcUfAfcuaaCfaUfcucacsusg 564 AD-520018.1 asUfsuuuAfgUfAfgucaAfgGfuuaucsasu 565 AD-520062.1 asUfsuuaGfuUfAfggugAfaAfaaggusgsu 566 AD-519754.1 asUfscauAfaAfAfccaaCfuCfagcucsasg 567 AD-520097.1 asAfsaauUfcAfAfcaggUfaAfcaacgscsu 568 AD-520352.1 asAfsaugAfaAfUfggugCfaAfucucasgsc 569 AD-519755.1 asUfsucaUfaAfAfaccaAfcUfcagcuscsa 570 AD-520063.1 asUfsuuuAfgUfUfagguGfaAfaaaggsusg 571 AD-520066.1 asUfsuauUfuUfAfguuaGfgUfgaaaasasg 572 AD-520068.1 asAfscauUfaUfUfuuagUfuAfggugasasa 573 AD-520465.1 asAfsaaaUfaAfGfccagGfuUfcaagususg 574 AD-519592.1 asUfsacuUfuAfUfugccCfaAfgaagasasg 575 AD-519591.1 asAfscuuUfaUfUfgcccAfaGfaagaasgsu 576 Duplex mRNA Target SEQ Name Sequence 5′to 3′ ID NO: AD-67605.6 UUACCUGUUGAAUUUUGUAUUAU 577 AD-520101.1 GUUACCUGUUGAAUUUUGUAUUA 578 AD-520098.1 GCGUUGUUACCUGUUGAAUUUUG 579 AD-67575.6 UGUUACCUGUUGAAUUUUGUAUU 580 AD-520467.1 ACUUGAACCUGGCUUAUUUUCUG 581 AD-520064.1 ACCUUUUUCACCUAACUAAAAUA 582 AD-520099.1 GUUGUUACCUGUUGAAUUUUGUA 583 AD-520466.1 AACUUGAACCUGGCUUAUUUUCU 584 AD-519351.1 UUAGGAUAAUGUCUUAUGUAAUG 585 AD-520065.1 CCUUUUUCACCUAACUAAAAUAA 586 AD-520069.1 UUCACCUAACUAAAAUAAUGUUU 587 AD-519828.1 UACAUCAGCAUGCGUUAAUUCAG 588 AD-520035.1 GGGGUAACAAGAUGAUAAUCUAC 589 AD-520067.1 UUUUUCACCUAACUAAAAUAAUG 590 AD-75289.2 UUUUCACCUAACUAAAAUAAUGU 591 AD-520125.1 CAGUGAGAUGUUAGUAGAAUAAG 592 AD-520018.1 AUGAUAACCUUGACUACUAAAAA 593 AD-520062.1 ACACCUUUUUCACCUAACUAAAA 594 AD-519754.1 CUGAGCUGAGUUGGUUUUAUGAA 595 AD-520097.1 AGCGUUGUUACCUGUUGAAUUUU 596 AD-520352.1 GCUGAGAUUGCACCAUUUCAUUC 597 AD-519755.1 UGAGCUGAGUUGGUUUUAUGAAA 598 AD-520063.1 CACCUUUUUCACCUAACUAAAAU 599 AD-520066.1 CUUUUUCACCUAACUAAAAUAAU 600 AD-520068.1 UUUCACCUAACUAAAAUAAUGUU 601 AD-520465.1 CAACUUGAACCUGGCUUAUUUUC 602 AD-519592.1 CUUCUUCUUGGGCAAUAAAGUAC 603 AD-519591.1 ACUUCUUCUUGGGCAAUAAAGUA 604

TABLE 6 Unmodified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ Duplex ID Range in ID Range in Name Sense Sequence 5′to 3′ NO: NM_025225.2 Antisense Sequence 5′to 3′ NO: NM_025225.2 AD-521420.1 GGAUAAUGUCUUAUGUAAU 605 1219-1237 AUUACAUAAGACAUUAUCC 712 1219-1237 AD-520973.1 GAGUGAGUGACAACGUACU 606 676-694 AGUACGUUGUCACUCACUC 713 676-694 AD-521124.1 GGAGAGAUAUGCCUUCGAU 607 876-894 AUCGAAGGCAUAUCUCUCC 714 876-894 AD-521486.1 UGGUGACAUGGCUUCCAGU 608 1288-1306 ACUGGAAGCCAUGUCACCA 715 1288-1306 AD-520903.1 GAAGUCGUGGAUGCCUUGU 609 582-600 ACAAGGCAUCCACGACUUC 716 582-600 AD-520972.1 GGAGUGAGUGACAACGUAU 610 675-693 AUACGUUGUCACUCACUCC 717 675-693 AD-521421.1 GAUAAUGUCUUAUGUAAUU 611 1220-1238 AAUUACAUAAGACAUUAUC 718 1220-1238 AD-521840.1 GGUUUUAUGAAAAGCUAGU 612 1753-1771 ACUAGCUUUUCAUAAAACC 719 1753-1771 AD-521003.1 CAAAACAACCAUCACCGUU 613 707-725 AACGGUGAUGGUUGUUUUG 720 707-725 AD-521129.1 GAUAUGCCUUCGAGGAUAU 614 881-899 AUAUCCUCGAAGGCAUAUC 721 881-899 AD-521465.1 CCAUUGCGAUUGUCCAGAU 615 1267-1285 AUCUGGACAAUCGCAAUGG 722 1267-1285 AD-521086.1 ACAGGGAACCUCUACCUUU 616 819-837 AAAGGUAGAGGUUCCCUGU 723 819-837 AD-521409.1 UGCUACCCAUUAGGAUAAU 617 1207-1225 AUUAUCCUAAUGGGUAGCA 724 1207-1225 AD-522178.1 CCUGUUGAAUUUUGUAUUU 618 2246-2264 AAAUACAAAAUUCAACAGG 725 2246-2264 AD-520974.1 AGUGAGUGACAACGUACCU 619 677-695 AGGUACGUUGUCACUCACU 726 677-695 AD-520902.1 CGAAGUCGUGGAUGCCUUU 620 581-599 AAAGGCAUCCACGACUUCG 727 581-599 AD-522140.1 CUUUUUCACCUAACUAAAU 621 2182-2200 AUUUAGUUAGGUGAAAAAG 728 2182-2200 AD-521410.1 GCUACCCAUUAGGAUAAUU 622 1208-1226 AAUUAUCCUAAUGGGUAGC 729 1208-1226 AD-522548.1 GAACCUGGCUUAUUUUCUU 623 2716-2734 AAGAAAAUAAGCCAGGUUC 730 2716-2734 AD-521002.1 CCAAAACAACCAUCACCGU 624 706-724 ACGGUGAUGGUUGUUUUGG 731 706-724 AD-522176.1 UUACCUGUUGAAUUUUGUU 625 2243-2261 AACAAAAUUCAACAGGUAA 732 2243-2261 AD-520926.1 CUACAGUGGCCUUAUCCCU 626 623-641 AGGGAUAAGGCCACUGUAG 733 623-641 AD-521895.1 CUAAUACAUCAGCAUGCGU 627 1811-1829 ACGCAUGCUGAUGUAUUAG 734 1811-1829 AD-521499.1 CCAGAUAUGCCCGACGAUU 628 1302-1320 AAUCGUCGGGCAUAUCUGG 735 1302-1320 AD-521466.1 CAUUGCGAUUGUCCAGAGU 629 1268-1286 ACUCUGGACAAUCGCAAUG 736 1268-1286 AD-521140.1 GAGGAUAUUUGGAUGCAUU 630 892-910 AAUGCAUCCAAAUAUCCUC 737 892-910 AD-520892.1 GGUCCAAAGACGAAGUCGU 631 571-589 ACGACUUCGUCUUUGGACC 738 571-589 AD-520976.1 UGAGUGACAACGUACCCUU 632 679-697 AAGGGUACGUUGUCACUCA 739 679-697 AD-521457.1 GGAAUCUGCCAUUGCGAUU 633 1259-1277 AAUCGCAAUGGCAGAUUCC 740 1259-1277 AD-521127.1 GAGAUAUGCCUUCGAGGAU 634 879-897 AUCCUCGAAGGCAUAUCUC 741 879-897 AD-522145.1 UCACCUAACUAAAAUAAUU 635 2187-2205 AAUUAUUUUAGUUAGGUGA 742 2187-2205 AD-520984.1 ACGUACCCUUCAUUGAUGU 636 688-706 ACAUCAAUGAAGGGUACGU 743 688-706 AD-521997.1 CUGUUCCAGCAUGAGGUUU 637 1916-1934 AAACCUCAUGCUGGAACAG 744 1916-1934 AD-522174.1 UGUUACCUGUUGAAUUUUU 638 2241-2259 AAAAAUUCAACAGGUAACA 745 2241-2259 AD-522545.1 UUGAACCUGGCUUAUUUUU 639 2714-2732 AAAAAUAAGCCAGGUUCAA 746 2714-2732 AD-521979.1 GGCCCUAUUAAUGGUCAGU 640 1897-1915 ACUGACCAUUAAUAGGGCC 747 1897-1915 AD-520891.1 CGGUCCAAAGACGAAGUCU 641 570-588 AGACUUCGUCUUUGGACCG 748 570-588 AD-521833.1 CUGAGUUGGUUUUAUGAAU 642 1746-1764 AUUCAUAAAACCAACUCAG 749 1746-1764 AD-521461.1 UCUGCCAUUGCGAUUGUCU 643 1263-1281 AGACAAUCGCAAUGGCAGA 750 1263-1281 AD-521386.1 GGAUACAUGAGCAAGAUUU 644 1182-1200 AAAUCUUGCUCAUGUAUCC 751 1182-1200 AD-521123.1 GGGAGAGAUAUGCCUUCGU 645 875-893 ACGAAGGCAUAUCUCUCCC 752 875-893 AD-520899.1 AGACGAAGUCGUGGAUGCU 646 578-596 AGCAUCCACGACUUCGUCU 753 578-596 AD-521089.1 GGGAACCUCUACCUUCUCU 647 822-840 AGAGAAGGUAGAGGUUCCC 754 822-840 AD-521407.1 CUUGCUACCCAUUAGGAUU 648 1205-1223 AAUCCUAAUGGGUAGCAAG 755 1205-1223 AD-520898.1 AAGACGAAGUCGUGGAUGU 649 577-595 ACAUCCACGACUUCGUCUU 756 577-595 AD-521378.1 ACAAAGGUGGAUACAUGAU 650 1174-1192 AUCAUGUAUCCACCUUUGU 757 1174-1192 AD-521500.1 CAGAUAUGCCCGACGAUGU 651 1303-1321 ACAUCGUCGGGCAUAUCUG 758 1303-1321 AD-521798.1 CAUUGCUGUGUAGUGACCU 652 1694-1712 AGGUCACUACACAGCAAUG 759 1694-1712 AD-521902.1 UCAGCAUGCGUUAAUUCAU 653 1819-1837 AUGAAUUAACGCAUGCUGA 760 1819-1837 AD-521896.1 UAAUACAUCAGCAUGCGUU 654 1812-1830 AACGCAUGCUGAUGUAUUA 761 1812-1830 AD-521989.1 AUGGUCAGACUGUUCCAGU 655 1907-1925 ACUGGAACAGUCUGACCAU 762 1907-1925 AD-520896.1 CAAAGACGAAGUCGUGGAU 656 575-593 AUCCACGACUUCGUCUUUG 763 575-593 AD-69024.2 CCUAACUAAAAUAAUGUUU 657 2190-2208 AAACAUUAUUUUAGUUAGG 764 2190-2208 AD-522146.1 CACCUAACUAAAAUAAUGU 658 2188-2206 ACAUUAUUUUAGUUAGGUG 765 2188-2206 AD-522432.1 AGAUUGCACCAUUUCAUUU 659 2546-2564 AAAUGAAAUGGUGCAAUCU 766 2546-2564 AD-521020.1 CUGCCCUAAAGUCAAGUCU 660 752-770 AGACUUGACUUUAGGGCAG 767 752-770 AD-521668.1 CUUCUUGGGCAAUAAAGUU 661 1541-1559 AACUUUAUUGCCCAAGAAG 768 1541-1559 AD-522097.1 UAACCUUGACUACUAAAAU 662 2112-2130 AUUUUAGUAGUCAAGGUUA 769 2112-2130 AD-520999.1 AUGCCAAAACAACCAUCAU 663 703-721 AUGAUGGUUGUUUUGGCAU 770 703-721 AD-521832.1 GCUGAGUUGGUUUUAUGAU 664 1745-1763 AUCAUAAAACCAACUCAGC 771 1745-1763 AD-520894.1 UCCAAAGACGAAGUCGUGU 665 573-591 ACACGACUUCGUCUUUGGA 772 573-591 AD-522144.1 UUCACCUAACUAAAAUAAU 666 2186-2204 AUUAUUUUAGUUAGGUGAA 773 2186-2204 AD-521408.1 UUGCUACCCAUUAGGAUAU 667 1206-1224 AUAUCCUAAUGGGUAGCAA 774 1206-1224 AD-521128.1 AGAUAUGCCUUCGAGGAUU 668 880-898 AAUCCUCGAAGGCAUAUCU 775 880-898 AD-521980.1 GCCCUAUUAAUGGUCAGAU 669 1898-1916 AUCUGACCAUUAAUAGGGC 776 1898-1916 AD-521406.1 ACUUGCUACCCAUUAGGAU 670 1204-1222 AUCCUAAUGGGUAGCAAGU 777 1204-1222 AD-521131.1 UAUGCCUUCGAGGAUAUUU 671 883-901 AAAUAUCCUCGAAGGCAUA 778 883-901 AD-521909.1 GCGUUAAUUCAGCUGGUUU 672 1826-1844 AAACCAGCUGAAUUAACGC 779 1826-1844 AD-521954.1 GAGGGUCCCUUACUGACUU 673 1872-1890 AAGUCAGUAAGGGACCCUC 780 1872-1890 AD-520872.1 CGUUCUGGUGUCUGACUUU 674 551-569 AAAGUCAGACACCAGAACG 781 551-569 AD-522142.1 UUUUCACCUAACUAAAAUU 675 2184-2202 AAUUUUAGUUAGGUGAAAA 782 2184-2202 AD-520994.1 CAUUGAUGCCAAAACAACU 676 698-716 AGUUGUUUUGGCAUCAAUG 783 698-716 AD-520886.1 ACUUUCGGUCCAAAGACGU 677 565-583 ACGUCUUUGGACCGAAAGU 784 565-583 AD-521987.1 UAAUGGUCAGACUGUUCCU 678 1905-1923 AGGAACAGUCUGACCAUUA 785 1905-1923 AD-521988.1 AAUGGUCAGACUGUUCCAU 679 1906-1924 AUGGAACAGUCUGACCAUU 786 1906-1924 AD-520785.1 AAGCAAGUUCCUCCGACAU 680 443-461 AUGUCGGAGGAACUUGCUU 787 443-461 AD-521919.1 AGCUGGUUGGGAAAUGACU 681 1836-1854 AGUCAUUUCCCAACCAGCU 788 1836-1854 AD-521121.1 CUGGGAGAGAUAUGCCUUU 682 873-891 AAAGGCAUAUCUCUCCCAG 789 873-891 AD-522202.1 GAGAUGUUAGUAGAAUAAU 683 2276-2294 AUUAUUCUACUAACAUCUC 790 2276-2294 AD-521130.1 AUAUGCCUUCGAGGAUAUU 684 882-900 AAUAUCCUCGAAGGCAUAU 791 882-900 AD-521908.1 UGCGUUAAUUCAGCUGGUU 685 1825-1843 AACCAGCUGAAUUAACGCA 792 1825-1843 AD-522173.1 UUGUUACCUGUUGAAUUUU 686 2240-2258 AAAAUUCAACAGGUAACAA 793 2240-2258 AD-521785.1 GCAGCUACCUCCGCAUUGU 687 1681-1699 ACAAUGCGGAGGUAGCUGC 794 1681-1699 AD-520890.1 UCGGUCCAAAGACGAAGUU 688 569-587 AACUUCGUCUUUGGACCGA 795 569-587 AD-520978.1 AGUGACAACGUACCCUUCU 689 681-699 AGAAGGGUACGUUGUCACU 796 681-699 AD-521744.1 GUCUAGCAGAUUCUUUCAU 690 1637-1655 AUGAAAGAAUCUGCUAGAC 797 1637-1655 AD-521021.1 UGCCCUAAAGUCAAGUCCU 691 753-771 AGGACUUGACUUUAGGGCA 798 753-771 AD-521197.1 UCCUCAGAAGGGAUGGAUU 692 966-984 AAUCCAUCCCUUCUGAGGA 799 966-984 AD-521379.1 CAAAGGUGGAUACAUGAGU 693 1175-1193 ACUCAUGUAUCCACCUUUG 800 1175-1193 AD-520925.1 UCUACAGUGGCCUUAUCCU 694 622-640 AGGAUAAGGCCACUGUAGA 801 622-640 AD-520888.1 UUUCGGUCCAAAGACGAAU 695 567-585 AUUCGUCUUUGGACCGAAA 802 567-585 AD-520975.1 GUGAGUGACAACGUACCCU 696 678-696 AGGGUACGUUGUCACUCAC 803 678-696 AD-521666.1 UUCUUCUUGGGCAAUAAAU 697 1539-1557 AUUUAUUGCCCAAGAAGAA 804 1539-1557 AD-521922.1 UGGUUGGGAAAUGACACCU 698 1839-1857 AGGUGUCAUUUCCCAACCA 805 1839-1857 AD-521986.1 UUAAUGGUCAGACUGUUCU 699 1904-1922 AGAACAGUCUGACCAUUAA 806 1904-1922 AD-521915.1 AUUCAGCUGGUUGGGAAAU 700 1832-1850 AUUUCCCAACCAGCUGAAU 807 1832-1850 AD-520897.1 AAAGACGAAGUCGUGGAUU 701 576-594 AAUCCACGACUUCGUCUUU 808 576-594 AD-520889.1 UUCGGUCCAAAGACGAAGU 702 568-586 ACUUCGUCUUUGGACCGAA 809 568-586 AD-520885.1 GACUUUCGGUCCAAAGACU 703 564-582 AGUCUUUGGACCGAAAGUC 810 564-582 AD-521469.1 UGCGAUUGUCCAGAGACUU 704 1271-1289 AAGUCUCUGGACAAUCGCA 811 1271-1289 AD-521674.1 GGGCAAUAAAGUACCUGCU 705 1547-1565 AGCAGGUACUUUAUUGCCC 812 1547-1565 AD-521983.1 CUAUUAAUGGUCAGACUGU 706 1901-1919 ACAGUCUGACCAUUAAUAG 813 1901-1919 AD-521385.1 UGGAUACAUGAGCAAGAUU 707 1181-1199 AAUCUUGCUCAUGUAUCCA 814 1181-1199 AD-521937.1 ACCAGGAAGCCCAGUGCAU 708 1854-1872 AUGCACUGGGCUUCCUGGU 815 1854-1872 AD-522141.1 UUUUUCACCUAACUAAAAU 709 2183-2201 AUUUUAGUUAGGUGAAAAA 816 2183-2201 AD-522143.1 UUUCACCUAACUAAAAUAU 710 2185-2203 AUAUUUUAGUUAGGUGAAA 817 2185-2203 AD-521669.1 UUCUUGGGCAAUAAAGUAU 711 1542-1560 AUACUUUAUUGCCCAAGAA 818 1542-1560

TABLE 7 Modified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents Duplex mRNA Target Name Sense Sequence 5′to 3′ SEQ ID NO: Antisense Sequence 5′to 3′ SEQ ID NO:  Sequence 5′to 3′ SEQ ID NO: AD-521420.1 GGAUAAUGUCUUAUGUAAUdTdT 819 AUUACAUAAGACAUUAUCCdTdT 926 GGAUAAUGUCUUAUGUAAU 1033 AD-520973.1 GAGUGAGUGACAACGUACUdTdT 820 AGUACGUUGUCACUCACUCdTdT 927 GAGUGAGUGACAACGUACC 1034 AD-521124.1 GGAGAGAUAUGCCUUCGAUdTdT 821 AUCGAAGGCAUAUCUCUCCdTdT 928 GGAGAGAUAUGCCUUCGAG 1035 AD-521486.1 UGGUGACAUGGCUUCCAGUdTdT 822 ACUGGAAGCCAUGUCACCAdTdT 929 UGGUGACAUGGCUUCCAGA 1036 AD-520903.1 GAAGUCGUGGAUGCCUUGUdTdT 823 ACAAGGCAUCCACGACUUCdTdT 930 GAAGUCGUGGAUGCCUUGG 1037 AD-520972.1 GGAGUGAGUGACAACGUAUdTdT 824 AUACGUUGUCACUCACUCCdTdT 931 GGAGUGAGUGACAACGUAC 1038 AD-521421.1 GAUAAUGUCUUAUGUAAUUdTdT 825 AAUUACAUAAGACAUUAUCdTdT 932 GAUAAUGUCUUAUGUAAUG 1039 AD-521840.1 GGUUUUAUGAAAAGCUAGUdTdT 826 ACUAGCUUUUCAUAAAACCdTdT 933 GGUUUUAUGAAAAGCUAGG 1040 AD-521003.1 CAAAACAACCAUCACCGUUdTdT 827 AACGGUGAUGGUUGUUUUGdTdT 934 CAAAACAACCAUCACCGUG 1041 AD-521129.1 GAUAUGCCUUCGAGGAUAUdTdT 828 AUAUCCUCGAAGGCAUAUCdTdT 935 GAUAUGCCUUCGAGGAUAU 1042 AD-521465.1 CCAUUGCGAUUGUCCAGAUdTdT 829 AUCUGGACAAUCGCAAUGGdTdT 936 CCAUUGCGAUUGUCCAGAG 1043 AD-521086.1 ACAGGGAACCUCUACCUUUdTdT 830 AAAGGUAGAGGUUCCCUGUdTdT 937 ACAGGGAACCUCUACCUUC 1044 AD-521409.1 UGCUACCCAUUAGGAUAAUdTdT 831 AUUAUCCUAAUGGGUAGCAdTdT 938 UGCUACCCAUUAGGAUAAU 1045 AD-522178.1 CCUGUUGAAUUUUGUAUUUdTdT 832 AAAUACAAAAUUCAACAGGdTdT 939 CCUGUUGAAUUUUGUAUUA 1046 AD-520974.1 AGUGAGUGACAACGUACCUdTdT 833 AGGUACGUUGUCACUCACUdTdT 940 AGUGAGUGACAACGUACCC 1047 AD-520902.1 CGAAGUCGUGGAUGCCUUUdTdT 834 AAAGGCAUCCACGACUUCGdTdT 941 CGAAGUCGUGGAUGCCUUG 1048 AD-522140.1 CUUUUUCACCUAACUAAAUdTdT 835 AUUUAGUUAGGUGAAAAAGdTdT 942 CUUUUUCACCUAACUAAAA 1049 AD-521410.1 GCUACCCAUUAGGAUAAUUdTdT 836 AAUUAUCCUAAUGGGUAGCdTdT 943 GCUACCCAUUAGGAUAAUG 1050 AD-522548.1 GAACCUGGCUUAUUUUCUUdTdT 837 AAGAAAAUAAGCCAGGUUCdTdT 944 GAACCUGGCUUAUUUUCUG 1051 AD-521002.1 CCAAAACAACCAUCACCGUdTdT 838 ACGGUGAUGGUUGUUUUGGdTdT 945 CCAAAACAACCAUCACCGU 1052 AD-522176.1 UUACCUGUUGAAUUUUGUUdTdT 839 AACAAAAUUCAACAGGUAAdTdT 946 UUACCUGUUGAAUUUUGUA 1053 AD-520926.1 CUACAGUGGCCUUAUCCCUdTdT 840 AGGGAUAAGGCCACUGUAGdTdT 947 CUACAGUGGCCUUAUCCCU 1054 AD-521895.1 CUAAUACAUCAGCAUGCGUdTdT 841 ACGCAUGCUGAUGUAUUAGdTdT 948 CUAAUACAUCAGCAUGCGU 1055 AD-521499.1 CCAGAUAUGCCCGACGAUUdTdT 842 AAUCGUCGGGCAUAUCUGGdTdT 949 CCAGAUAUGCCCGACGAUG 1056 AD-521466.1 CAUUGCGAUUGUCCAGAGUdTdT 843 ACUCUGGACAAUCGCAAUGdTdT 950 CAUUGCGAUUGUCCAGAGA 1057 AD-521140.1 GAGGAUAUUUGGAUGCAUUdTdT 844 AAUGCAUCCAAAUAUCCUCdTdT 951 GAGGAUAUUUGGAUGCAUU 1058 AD-520892.1 GGUCCAAAGACGAAGUCGUdTdT 845 ACGACUUCGUCUUUGGACCdTdT 952 GGUCCAAAGACGAAGUCGU 1059 AD-520976.1 UGAGUGACAACGUACCCUUdTdT 846 AAGGGUACGUUGUCACUCAdTdT 953 UGAGUGACAACGUACCCUU 1060 AD-521457.1 GGAAUCUGCCAUUGCGAUUdTdT 847 AAUCGCAAUGGCAGAUUCCdTdT 954 GGAAUCUGCCAUUGCGAUU 1061 AD-521127.1 GAGAUAUGCCUUCGAGGAUdTdT 848 AUCCUCGAAGGCAUAUCUCdTdT 955 GAGAUAUGCCUUCGAGGAU 1062 AD-522145.1 UCACCUAACUAAAAUAAUUdTdT 849 AAUUAUUUUAGUUAGGUGAdTdT 956 UCACCUAACUAAAAUAAUG 1063 AD-520984.1 ACGUACCCUUCAUUGAUGUdTdT 850 ACAUCAAUGAAGGGUACGUdTdT 957 ACGUACCCUUCAUUGAUGC 1064 AD-521997.1 CUGUUCCAGCAUGAGGUUUdTdT 851 AAACCUCAUGCUGGAACAGdTdT 958 CUGUUCCAGCAUGAGGUUC 1065 AD-522174.1 UGUUACCUGUUGAAUUUUUdTdT 852 AAAAAUUCAACAGGUAACAdTdT 959 UGUUACCUGUUGAAUUUUG 1066 AD-522545.1 UUGAACCUGGCUUAUUUUUdTdT 853 AAAAAUAAGCCAGGUUCAAdTdT 960 UUGAACCUGGCUUAUUUUC 1067 AD-521979.1 GGCCCUAUUAAUGGUCAGUdTdT 854 ACUGACCAUUAAUAGGGCCdTdT 961 GGCCCUAUUAAUGGUCAGA 1068 AD-520891.1 CGGUCCAAAGACGAAGUCUdTdT 855 AGACUUCGUCUUUGGACCGdTdT 962 CGGUCCAAAGACGAAGUCG 1069 AD-521833.1 CUGAGUUGGUUUUAUGAAUdTdT 856 AUUCAUAAAACCAACUCAGdTdT 963 CUGAGUUGGUUUUAUGAAA 1070 AD-521461.1 UCUGCCAUUGCGAUUGUCUdTdT 857 AGACAAUCGCAAUGGCAGAdTdT 964 UCUGCCAUUGCGAUUGUCC 1071 AD-521386.1 GGAUACAUGAGCAAGAUUUdTdT 858 AAAUCUUGCUCAUGUAUCCdTdT 965 GGAUACAUGAGCAAGAUUU 1072 AD-521123.1 GGGAGAGAUAUGCCUUCGUdTdT 859 ACGAAGGCAUAUCUCUCCCdTdT 966 GGGAGAGAUAUGCCUUCGA 1073 AD-520899.1 AGACGAAGUCGUGGAUGCUdTdT 860 AGCAUCCACGACUUCGUCUdTdT 967 AGACGAAGUCGUGGAUGCC 1074 AD-521089.1 GGGAACCUCUACCUUCUCUdTdT 861 AGAGAAGGUAGAGGUUCCCdTdT 968 GGGAACCUCUACCUUCUCU 1075 AD-521407.1 CUUGCUACCCAUUAGGAUUdTdT 862 AAUCCUAAUGGGUAGCAAGdTdT 969 CUUGCUACCCAUUAGGAUA 1076 AD-520898.1 AAGACGAAGUCGUGGAUGUdTdT 863 ACAUCCACGACUUCGUCUUdTdT 970 AAGACGAAGUCGUGGAUGC 1077 AD-521378.1 ACAAAGGUGGAUACAUGAUdTdT 864 AUCAUGUAUCCACCUUUGUdTdT 971 ACAAAGGUGGAUACAUGAG 1078 AD-521500.1 CAGAUAUGCCCGACGAUGUdTdT 865 ACAUCGUCGGGCAUAUCUGdTdT 972 CAGAUAUGCCCGACGAUGU 1079 AD-521798.1 CAUUGCUGUGUAGUGACCUdTdT 866 AGGUCACUACACAGCAAUGdTdT 973 CAUUGCUGUGUAGUGACCC 1080 AD-521902.1 UCAGCAUGCGUUAAUUCAUdTdT 867 AUGAAUUAACGCAUGCUGAdTdT 974 UCAGCAUGCGUUAAUUCAG 1081 AD-521896.1 UAAUACAUCAGCAUGCGUUdTdT 868 AACGCAUGCUGAUGUAUUAdTdT 975 UAAUACAUCAGCAUGCGUU 1082 AD-521989.1 AUGGUCAGACUGUUCCAGUdTdT 869 ACUGGAACAGUCUGACCAUdTdT 976 AUGGUCAGACUGUUCCAGC 1083 AD-520896.1 CAAAGACGAAGUCGUGGAUdTdT 870 AUCCACGACUUCGUCUUUGdTdT 977 CAAAGACGAAGUCGUGGAU 1084 AD-69024.2 CCUAACUAAAAUAAUGUUUdTdT 871 AAACAUUAUUUUAGUUAGGdTdT 978 CCUAACUAAAAUAAUGUUU 1085 AD-522146.1 CACCUAACUAAAAUAAUGUdTdT 872 ACAUUAUUUUAGUUAGGUGdTdT 979 CACCUAACUAAAAUAAUGU 1086 AD-522432.1 AGAUUGCACCAUUUCAUUUdTdT 873 AAAUGAAAUGGUGCAAUCUdTdT 980 AGAUUGCACCAUUUCAUUC 1087 AD-521020.1 CUGCCCUAAAGUCAAGUCUdTdT 874 AGACUUGACUUUAGGGCAGdTdT 981 CUGCCCUAAAGUCAAGUCC 1088 AD-521668.1 CUUCUUGGGCAAUAAAGUUdTdT 875 AACUUUAUUGCCCAAGAAGdTdT 982 CUUCUUGGGCAAUAAAGUA 1089 AD-522097.1 UAACCUUGACUACUAAAAUdTdT 876 AUUUUAGUAGUCAAGGUUAdTdT 983 UAACCUUGACUACUAAAAA 1090 AD-520999.1 AUGCCAAAACAACCAUCAUdTdT 877 AUGAUGGUUGUUUUGGCAUdTdT 984 AUGCCAAAACAACCAUCAC 1091 AD-521832.1 GCUGAGUUGGUUUUAUGAUdTdT 878 AUCAUAAAACCAACUCAGCdTdT 985 GCUGAGUUGGUUUUAUGAA 1092 AD-520894.1 UCCAAAGACGAAGUCGUGUdTdT 879 ACACGACUUCGUCUUUGGAdTdT 986 UCCAAAGACGAAGUCGUGG 1093 AD-522144.1 UUCACCUAACUAAAAUAAUdTdT 880 AUUAUUUUAGUUAGGUGAAdTdT 987 UUCACCUAACUAAAAUAAU 1094 AD-521408.1 UUGCUACCCAUUAGGAUAUdTdT 881 AUAUCCUAAUGGGUAGCAAdTdT 988 UUGCUACCCAUUAGGAUAA 1095 AD-521128.1 AGAUAUGCCUUCGAGGAUUdTdT 882 AAUCCUCGAAGGCAUAUCUdTdT 989 AGAUAUGCCUUCGAGGAUA 1096 AD-521980.1 GCCCUAUUAAUGGUCAGAUdTdT 883 AUCUGACCAUUAAUAGGGCdTdT 990 GCCCUAUUAAUGGUCAGAC 1097 AD-521406.1 ACUUGCUACCCAUUAGGAUdTdT 884 AUCCUAAUGGGUAGCAAGUdTdT 991 ACUUGCUACCCAUUAGGAU 1098 AD-521131.1 UAUGCCUUCGAGGAUAUUUdTdT 885 AAAUAUCCUCGAAGGCAUAdTdT 992 UAUGCCUUCGAGGAUAUUU 1099 AD-521909.1 GCGUUAAUUCAGCUGGUUUdTdT 886 AAACCAGCUGAAUUAACGCdTdT 993 GCGUUAAUUCAGCUGGUUG 1100 AD-521954.1 GAGGGUCCCUUACUGACUUdTdT 887 AAGUCAGUAAGGGACCCUCdTdT 994 GAGGGUCCCUUACUGACUG 1101 AD-520872.1 CGUUCUGGUGUCUGACUUUdTdT 888 AAAGUCAGACACCAGAACGdTdT 995 CGUUCUGGUGUCUGACUUU 1102 AD-522142.1 UUUUCACCUAACUAAAAUUdTdT 889 AAUUUUAGUUAGGUGAAAAdTdT 996 UUUUCACCUAACUAAAAUA 1103 AD-520994.1 CAUUGAUGCCAAAACAACUdTdT 890 AGUUGUUUUGGCAUCAAUGdTdT 997 CAUUGAUGCCAAAACAACC 1104 AD-520886.1 ACUUUCGGUCCAAAGACGUdTdT 891 ACGUCUUUGGACCGAAAGUdTdT 998 ACUUUCGGUCCAAAGACGA 1105 AD-521987.1 UAAUGGUCAGACUGUUCCUdTdT 892 AGGAACAGUCUGACCAUUAdTdT 999 UAAUGGUCAGACUGUUCCA 1106 AD-521988.1 AAUGGUCAGACUGUUCCAUdTdT 893 AUGGAACAGUCUGACCAUUdTdT 1000 AAUGGUCAGACUGUUCCAG 1107 AD-520785.1 AAGCAAGUUCCUCCGACAUdTdT 894 AUGUCGGAGGAACUUGCUUdTdT 1001 AAGCAAGUUCCUCCGACAG 1108 AD-521919.1 AGCUGGUUGGGAAAUGACUdTdT 895 AGUCAUUUCCCAACCAGCUdTdT 1002 AGCUGGUUGGGAAAUGACA 1109 AD-521121.1 CUGGGAGAGAUAUGCCUUUdTdT 896 AAAGGCAUAUCUCUCCCAGdTdT 1003 CUGGGAGAGAUAUGCCUUC 1110 AD-522202.1 GAGAUGUUAGUAGAAUAAUdTdT 897 AUUAUUCUACUAACAUCUCdTdT 1004 GAGAUGUUAGUAGAAUAAG 1111 AD-521130.1 AUAUGCCUUCGAGGAUAUUdTdT 898 AAUAUCCUCGAAGGCAUAUdTdT 1005 AUAUGCCUUCGAGGAUAUU 1112 AD-521908.1 UGCGUUAAUUCAGCUGGUUdTdT 899 AACCAGCUGAAUUAACGCAdTdT 1006 UGCGUUAAUUCAGCUGGUU 1113 AD-522173.1 UUGUUACCUGUUGAAUUUUdTdT 900 AAAAUUCAACAGGUAACAAdTdT 1007 UUGUUACCUGUUGAAUUUU 1114 AD-521785.1 GCAGCUACCUCCGCAUUGUdTdT 901 ACAAUGCGGAGGUAGCUGCdTdT 1008 GCAGCUACCUCCGCAUUGC 1115 AD-520890.1 UCGGUCCAAAGACGAAGUUdTdT 902 AACUUCGUCUUUGGACCGAdTdT 1009 UCGGUCCAAAGACGAAGUC 1116 AD-520978.1 AGUGACAACGUACCCUUCUdTdT 903 AGAAGGGUACGUUGUCACUdTdT 1010 AGUGACAACGUACCCUUCA 1117 AD-521744.1 GUCUAGCAGAUUCUUUCAUdTdT 904 AUGAAAGAAUCUGCUAGACdTdT 1011 GUCUAGCAGAUUCUUUCAG 1118 AD-521021.1 UGCCCUAAAGUCAAGUCCUdTdT 905 AGGACUUGACUUUAGGGCAdTdT 1012 UGCCCUAAAGUCAAGUCCA 1119 AD-521197.1 UCCUCAGAAGGGAUGGAUUdTdT 906 AAUCCAUCCCUUCUGAGGAdTdT 1013 UCCUCAGAAGGGAUGGAUC 1120 AD-521379.1 CAAAGGUGGAUACAUGAGUdTdT 907 ACUCAUGUAUCCACCUUUGdTdT 1014 CAAAGGUGGAUACAUGAGC 1121 AD-520925.1 UCUACAGUGGCCUUAUCCUdTdT 908 AGGAUAAGGCCACUGUAGAdTdT 1015 UCUACAGUGGCCUUAUCCC 1122 AD-520888.1 UUUCGGUCCAAAGACGAAUdTdT 909 AUUCGUCUUUGGACCGAAAdTdT 1016 UUUCGGUCCAAAGACGAAG 1123 AD-520975.1 GUGAGUGACAACGUACCCUdTdT 910 AGGGUACGUUGUCACUCACdTdT 1017 GUGAGUGACAACGUACCCU 1124 AD-521666.1 UUCUUCUUGGGCAAUAAAUdTdT 911 AUUUAUUGCCCAAGAAGAAdTdT 1018 UUCUUCUUGGGCAAUAAAG 1125 AD-521922.1 UGGUUGGGAAAUGACACCUdTdT 912 AGGUGUCAUUUCCCAACCAdTdT 1019 UGGUUGGGAAAUGACACCA 1126 AD-521986.1 UUAAUGGUCAGACUGUUCUdTdT 913 AGAACAGUCUGACCAUUAAdTdT 1020 UUAAUGGUCAGACUGUUCC 1127 AD-521915.1 AUUCAGCUGGUUGGGAAAUdTdT 914 AUUUCCCAACCAGCUGAAUdTdT 1021 AUUCAGCUGGUUGGGAAAU 1128 AD-520897.1 AAAGACGAAGUCGUGGAUUdTdT 915 AAUCCACGACUUCGUCUUUdTdT 1022 AAAGACGAAGUCGUGGAUG 1129 AD-520889.1 UUCGGUCCAAAGACGAAGUdTdT 916 ACUUCGUCUUUGGACCGAAdTdT 1023 UUCGGUCCAAAGACGAAGU 1130 AD-520885.1 GACUUUCGGUCCAAAGACUdTdT 917 AGUCUUUGGACCGAAAGUCdTdT 1024 GACUUUCGGUCCAAAGACG 1131 AD-521469.1 UGCGAUUGUCCAGAGACUUdTdT 918 AAGUCUCUGGACAAUCGCAdTdT 1025 UGCGAUUGUCCAGAGACUG 1132 AD-521674.1 GGGCAAUAAAGUACCUGCUdTdT 919 AGCAGGUACUUUAUUGCCCdTdT 1026 GGGCAAUAAAGUACCUGCU 1133 AD-521983.1 CUAUUAAUGGUCAGACUGUdTdT 920 ACAGUCUGACCAUUAAUAGdTdT 1027 CUAUUAAUGGUCAGACUGU 1134 AD-521385.1 UGGAUACAUGAGCAAGAUUdTdT 921 AAUCUUGCUCAUGUAUCCAdTdT 1028 UGGAUACAUGAGCAAGAUU 1135 AD-521937.1 ACCAGGAAGCCCAGUGCAUdTdT 922 AUGCACUGGGCUUCCUGGUdTdT 1029 ACCAGGAAGCCCAGUGCAG 1136 AD-522141.1 UUUUUCACCUAACUAAAAUdTdT 923 AUUUUAGUUAGGUGAAAAAdTdT 1030 UUUUUCACCUAACUAAAAU 1137 AD-522143.1 UUUCACCUAACUAAAAUAUdTdT 924 AUAUUUUAGUUAGGUGAAAdTdT 1031 UUUCACCUAACUAAAAUAA 1138 AD-521669.1 UUCUUGGGCAAUAAAGUAUdTdT 925 AUACUUUAUUGCCCAAGAAdTdT 1032 UUCUUGGGCAAUAAAGUAC 1139

TABLE 8 Unmodified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ ID Range in ID Range in Duplex Name Sense Sequence 5′to 3′ NO: NM_025225.2 Antisense Sequence 5′to 3′ NO: NM_025225.2 AD-520062.3 ACCUUUUUCACCUAACUAAAU 1140 2180-2200 AUUUAGUUAGGUGAAAAAGGUGU 1230 2178-2200 AD-520060.1 ACACCUUUUUCACCUAACUAU 1141 2178-2198 AUAGUUAGGUGAAAAAGGUGUUC 1231 2176-2198 AD-520064.3 CUUUUUCACCUAACUAAAAUU 1142 2182-2202 AAUUUUAGUUAGGUGAAAAAGGU 1232 2180-2202 AD-518983.1 ACUUUCUUCAUGUGGACAUCU 1143 775-795 AGAUGUCCACAUGAAGAAAGUUC 1233 773-795 AD-520061.1 CACCUUUUUCACCUAACUAAU 1144 2179-2199 AUUAGUUAGGUGAAAAAGGUGUU 1234 2177-2199 AD-520063.2 CCUUUUUCACCUAACUAAAAU 1145 2181-2201 AUUUUAGUUAGGUGAAAAAGGUG 1235 2179-2201 AD-519615.1 UCCACCUUUCCCAGUUUUUCU 1146 1581-1601 AGAAAAACUGGGAAAGGUGGAGA 1236 1579-1601 AD-519757.1 CUGAGUUGGUUUUAUGAAAAU 1147 1746-1766 AUUUUCAUAAAACCAACUCAGCU 1237 1744-1766 AD-519329.1 AAGAUUUGCAACUUGCUACCU 1148 1194-1214 AGGUAGCAAGUUGCAAAUCUUGC 1238 1192-1214 AD-519324.1 UGAGCAAGAUUUGCAACUUGU 1149 1189-1209 ACAAGUUGCAAAUCUUGCUCAUG 1239 1187-1209 AD-518811.1 AAACGUUCUGGUGUCUGACUU 1150 548-568 AAGUCAGACACCAGAACGUUUUC 1240 546-568 AD-520059.1 AACACCUUUUUCACCUAACUU 1151 2177-2197 AAGUUAGGUGAAAAAGGUGUUCU 1241 2175-2197 AD-519616.1 CCACCUUUCCCAGUUUUUCAU 1152 1582-1602 AUGAAAAACUGGGAAAGGUGGAG 1242 1580-1602 AD-518655.1 CAGGUCCUCUCAGAUCUUGUU 1153 372-392 AACAAGAUCUGAGAGGACCUGCA 1243 370-392 AD-519617.1 CACCUUUCCCAGUUUUUCACU 1154 1583-1603 AGUGAAAAACUGGGAAAGGUGGA 1244 1581-1603 AD-519307.1 AAAGACAAAGGUGGAUACAUU 1155 1170-1190 AAUGUAUCCACCUUUGUCUUUCA 1245 1168-1190 AD-520065.3 UUUUUCACCUAACUAAAAUAU 1156 2183-2203 AUAUUUUAGUUAGGUGAAAAAGG 1246 2181-2203 AD-519323.1 AUGAGCAAGAUUUGCAACUUU 1157 1188-1208 AAAGUUGCAAAUCUUGCUCAUGU 1247 1186-1208 AD-519331.1 AUUUGCAACUUGCUACCCAUU 1158 1197-1217 AAUGGGUAGCAAGUUGCAAAUCU 1248 1195-1217 AD-518922.1 GACAACGUACCCUUCAUUGAU 1159 684-704 AUCAAUGAAGGGUACGUUGUCAC 1249 682-704 AD-519339.1 UUGCUACCCAUUAGGAUAAUU 1160 1206-1226 AAUUAUCCUAAUGGGUAGCAAGU 1250 1204-1226 AD-67552.2 UGCCUUCGAGGAUAUUUGGAU 1161 885-905 AUCCAAAUAUCCUCGAAGGCAUA 1251 883-905 AD-519756.1 GCUGAGUUGGUUUUAUGAAAU 1162 1745-1765 AUUUCAUAAAACCAACUCAGCUC 1252 1743-1765 AD-519333.1 UUGCAACUUGCUACCCAUUAU 1163 1199-1219 AUAAUGGGUAGCAAGUUGCAAAU 1253 1197-1219 AD-519612.1 CUCUCCACCUUUCCCAGUUUU 1164 1578-1598 AAAACUGGGAAAGGUGGAGAGCC 1254 1576-1598 AD-519762.1 UUGGUUUUAUGAAAAGCUAGU 1165 1751-1771 ACUAGCUUUUCAUAAAACCAACU 1255 1749-1771 AD-75265.3 CAUGAGCAAGAUUUGCAACUU 1166 1187-1207 AAGUUGCAAAUCUUGCUCAUGUA 1256 1185-1207 AD-67554.5 UCUGAGCUGAGUUGGUUUUAU 1167 1740-1760 AUAAAACCAACUCAGCUCAGAGG 1257 1738-1760 AD-518928.1 GUACCCUUCAUUGAUGCCAAU 1168 690-710 AUUGGCAUCAAUGAAGGGUACGU 1258 688-710 AD-519578.1 GUCCAGCCUGAACUUCUUCUU 1169 1526-1546 AAGAAGAAGUUCAGGCUGGACCU 1259 1524-1546 AD-75277.3 CUUGCUACCCAUUAGGAUAAU 1170 1205-1225 AUUAUCCUAAUGGGUAGCAAGUU 1260 1203-1225 AD-519317.1 UGGAUACAUGAGCAAGAUUUU 1171 1181-1201 AAAAUCUUGCUCAUGUAUCCACC 1261 1179-1201 AD-67581.5 CCUAUUAAUGGUCAGACUGUU 1172 1900-1920 AACAGUCUGACCAUUAAUAGGGC 1262 1898-1920 AD-519753.1 UGAGCUGAGUUGGUUUUAUGU 1173 1742-1762 ACAUAAAACCAACUCAGCUCAGA 1263 1740-1762 AD-518701.1 AUCUUCCAUCCAUCCUUCAAU 1174 420-440 AUUGAAGGAUGGAUGGAAGAUGC 1264 418-440 AD-519752.1 CUGAGCUGAGUUGGUUUUAUU 1175 1741-1761 AAUAAAACCAACUCAGCUCAGAG 1265 1739-1761 AD-519328.1 CAAGAUUUGCAACUUGCUACU 1176 1193-1213 AGUAGCAAGUUGCAAAUCUUGCU 1266 1191-1213 AD-519322.1 ACAUGAGCAAGAUUUGCAACU 1177 1186-1206 AGUUGCAAAUCUUGCUCAUGUAU 1267 1184-1206 AD-519911.1 CUAUUAAUGGUCAGACUGUUU 1178 1901-1921 AAACAGUCUGACCAUUAAUAGGG 1268 1899-1921 AD-519029.1 AGGGAACCUCUACCUUCUCUU 1179 821-841 AAGAGAAGGUAGAGGUUCCCUGU 1269 819-841 AD-519913.1 AUUAAUGGUCAGACUGUUCCU 1180 1903-1923 AGGAACAGUCUGACCAUUAAUAG 1270 1901-1923 AD-518924.1 CAACGUACCCUUCAUUGAUGU 1181 686-706 ACAUCAAUGAAGGGUACGUUGUC 1271 684-706 AD-519766.1 UUUUAUGAAAAGCUAGGAAGU 1182 1755-1775 ACUUCCUAGCUUUUCAUAAAACC 1272 1753-1775 AD-519069.1 UAUGCCUUCGAGGAUAUUUGU 1183 883-903 ACAAAUAUCCUCGAAGGCAUAUC 1273 881-903 AD-519614.1 CUCCACCUUUCCCAGUUUUUU 1184 1580-1600 AAAAAACUGGGAAAGGUGGAGAG 1274 1578-1600 AD-519618.1 ACCUUUCCCAGUUUUUCACUU 1185 1584-1604 AAGUGAAAAACUGGGAAAGGUGG 1275 1582-1604 AD-519326.1 AGCAAGAUUUGCAACUUGCUU 1186 1191-1211 AAGCAAGUUGCAAAUCUUGCUCA 1276 1189-1211 AD-518920.1 GUGACAACGUACCCUUCAUUU 1187 682-702 AAAUGAAGGGUACGUUGUCACUC 1277 680-702 AD-519760.1 AGUUGGUUUUAUGAAAAGCUU 1188 1749-1769 AAGCUUUUCAUAAAACCAACUCA 1278 1747-1769 AD-518813.1 CGUUCUGGUGUCUGACUUUCU 1189 551-571 AGAAAGUCAGACACCAGAACGUU 1279 549-571 AD-519396.1 UCUGCCAUUGCGAUUGUCCAU 1190 1263-1283 AUGGACAAUCGCAAUGGCAGAUU 1280 1261-1283 AD-519935.1 CAUGAGGUUCUUAGAAUGACU 1191 1925-1945 AGUCAUUCUAAGAACCUCAUGCU 1281 1923-1945 AD-519758.1 UGAGUUGGUUUUAUGAAAAGU 1192 1747-1767 ACUUUUCAUAAAACCAACUCAGC 1282 1745-1767 AD-518831.1 CGGUCCAAAGACGAAGUCGUU 1193 570-590 AACGACUUCGUCUUUGGACCGAA 1283 568-590 AD-518923.1 ACAACGUACCCUUCAUUGAUU 1194 685-705 AAUCAAUGAAGGGUACGUUGUCA 1284 683-705 AD-519755.2 AGCUGAGUUGGUUUUAUGAAU 1195 1744-1764 AUUCAUAAAACCAACUCAGCUCA 1285 1742-1764 AD-520116.1 UGUGAAUCAGUGAGAUGUUAU 1196 2265-2285 AUAACAUCUCACUGAUUCACAUA 1286 2263-2285 AD-519093.1 AUUCAGGUUCUUGGAAGAGAU 1197 908-928 AUCUCUUCCAAGAACCUGAAUGC 1287 906-928 AD-67588.2 AACGUUCUGGUGUCUGACUUU 1198 549-569 AAAGUCAGACACCAGAACGUUUU 1288 547-569 AD-519754.3 GAGCUGAGUUGGUUUUAUGAU 1199 1743-1763 AUCAUAAAACCAACUCAGCUCAG 1289 1741-1763 AD-519308.1 AAGACAAAGGUGGAUACAUGU 1200 1171-1191 ACAUGUAUCCACCUUUGUCUUUC 1290 1169-1191 AD-519759.1 GAGUUGGUUUUAUGAAAAGCU 1201 1748-1768 AGCUUUUCAUAAAACCAACUCAG 1291 1746-1768 AD-519763.1 UGGUUUUAUGAAAAGCUAGGU 1202 1752-1772 ACCUAGCUUUUCAUAAAACCAAC 1292 1750-1772 AD-519327.1 GCAAGAUUUGCAACUUGCUAU 1203 1192-1212 AUAGCAAGUUGCAAAUCUUGCUC 1293 1190-1212 AD-519761.1 GUUGGUUUUAUGAAAAGCUAU 1204 1750-1770 AUAGCUUUUCAUAAAACCAACUC 1294 1748-1770 AD-518702.1 UCUUCCAUCCAUCCUUCAACU 1205 421-441 AGUUGAAGGAUGGAUGGAAGAUG 1295 419-441 AD-519094.1 UUCAGGUUCUUGGAAGAGAAU 1206 909-929 AUUCUCUUCCAAGAACCUGAAUG 1296 907-929 AD-518832.1 GGUCCAAAGACGAAGUCGUGU 1207 571-591 ACACGACUUCGUCUUUGGACCGA 1297 569-591 AD-519767.1 UUUAUGAAAAGCUAGGAAGCU 1208 1756-1776 AGCUUCCUAGCUUUUCAUAAAAC 1298 1754-1776 AD-518917.1 UGAGUGACAACGUACCCUUCU 1209 679-699 AGAAGGGUACGUUGUCACUCACU 1299 677-699 AD-518988.1 CUUCAUGUGGACAUCACCAAU 1210 780-800 AUUGGUGAUGUCCACAUGAAGAA 1300 778-800 AD-519305.1 UGAAAGACAAAGGUGGAUACU 1211 1168-1188 AGUAUCCACCUUUGUCUUUCAUU 1301 1166-1188 AD-519613.1 UCUCCACCUUUCCCAGUUUUU 1212 1579-1599 AAAAACUGGGAAAGGUGGAGAGC 1302 1577-1599 AD-519121.1 CCUGAAGUCAUCCUCAGAAGU 1213 956-976 ACUUCUGAGGAUGACUUCAGGCC 1303 954-976 AD-519095.1 UCAGGUUCUUGGAAGAGAAGU 1214 910-930 ACUUCUCUUCCAAGAACCUGAAU 1304 908-930 AD-518982.1 AACUUUCUUCAUGUGGACAUU 1215 774-794 AAUGUCCACAUGAAGAAAGUUCG 1305 772-794 AD-519096.1 CAGGUUCUUGGAAGAGAAGGU 1216 911-931 ACCUUCUCUUCCAAGAACCUGAA 1306 909-931 AD-518986.1 UUCUUCAUGUGGACAUCACCU 1217 778-798 AGGUGAUGUCCACAUGAAGAAAG 1307 776-798 AD-518921.1 UGACAACGUACCCUUCAUUGU 1218 683-703 ACAAUGAAGGGUACGUUGUCACU 1308 681-703 AD-518985.1 UUUCUUCAUGUGGACAUCACU 1219 777-797 AGUGAUGUCCACAUGAAGAAAGU 1309 775-797 AD-518984.1 CUUUCUUCAUGUGGACAUCAU 1220 776-796 AUGAUGUCCACAUGAAGAAAGUU 1310 774-796 AD-519325.1 GAGCAAGAUUUGCAACUUGCU 1221 1190-1210 AGCAAGUUGCAAAUCUUGCUCAU 1311 1188-1210 AD-518812.1 ACGUUCUGGUGUCUGACUUUU 1222 550-570 AAAAGUCAGACACCAGAACGUUU 1312 548-570 AD-518987.1 UCUUCAUGUGGACAUCACCAU 1223 779-799 AUGGUGAUGUCCACAUGAAGAAA 1313 777-799 AD-518958.1 CAUCUGCCCUAAAGUCAAGUU 1224 749-769 AACUUGACUUUAGGGCAGAUGUC 1314 747-769 AD-67572.2 UUCUUUCAGAGGUGCUAAAGU 1225 1647-1667 ACUUUAGCACCUCUGAAAGAAUC 1315 1645-1667 AD-518980.1 CGAACUUUCUUCAUGUGGACU 1226 772-792 AGUCCACAUGAAGAAAGUUCGUG 1316 770-792 AD-519070.1 AUGCCUUCGAGGAUAUUUGGU 1227 884-904 ACCAAAUAUCCUCGAAGGCAUAU 1317 882-904 AD-518925.1 AACGUACCCUUCAUUGAUGCU 1228 687-707 AGCAUCAAUGAAGGGUACGUUGU 1318 685-707 AD-518981.1 GAACUUUCUUCAUGUGGACAU 1229 773-793 AUGUCCACAUGAAGAAAGUUCGU 1319 771-793

TABLE 9 Modified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ SEQ ID ID mRNA Target ID Duplex Name Sense Sequnce 5′to 3′ NO: Antisense Sequence 5′to 3′ NO: Sequence 5′to 3′ NO: AD-520062.3 ascscuuuUfuCfAfCfcuaacuaaauL96 1320 asUfsuuaGfuUfAfggugAfaAfaaggusgsu 1410 ACACCUUUUUCACCUAACUAAAA 1500 AD-520060.1 ascsaccuUfuUfUfCfaccuaacuauL96 1321 asUfsaguUfaGfGfugaaAfaAfggugususc 1411 GAACACCUUUUUCACCUAACUAA 1501 AD-520064.3 csusuuuuCfaCfCfUfaacuaaaauuL96 1322 asAfsuuuUfaGfUfuaggUfgAfaaaagsgsu 1412 ACCUUUUUCACCUAACUAAAAUA 1502 AD-518983.1 ascsuuucUfuCfAfUfguggacaucuL96 1323 asGfsaugUfcCfAfcaugAfaGfaaagususc 1413 GAACUUUCUUCAUGUGGACAUCA 1503 AD-520061.1 csasccuuUfuUfCfAfccuaacuaauL96 1324 asUfsuagUfuAfGfgugaAfaAfaggugsusu 1414 AACACCUUUUUCACCUAACUAAA 1504 AD-520063.2 cscsuuuuUfcAfCfCfuaacuaaaauL96 1325 asUfsuuuAfgUfUfagguGfaAfaaaggsusg 1415 CACCUUUUUCACCUAACUAAAAU 1505 AD-519615.1 uscscaccUfuUfCfCfcaguuuuucuL96 1326 asGfsaaaAfaCfUfgggaAfaGfguggasgsa 1416 UCUCCACCUUUCCCAGUUUUUCA 1506 AD-519757.1 csusgaguUfgGfUfUfuuaugaaaauL96 1327 asUfsuuuCfaUfAfaaacCfaAfcucagscsu 1417 AGCUGAGUUGGUUUUAUGAAAAG 1507 AD-519329.1 asasgauuUfgCfAfAfcuugcuaccuL96 1328 asGfsguaGfcAfAfguugCfaAfaucuusgsc 1418 GCAAGAUUUGCAACUUGCUACCC 1508 AD-519324.1 usgsagcaAfgAfUfUfugcaacuuguL96 1329 asCfsaagUfuGfCfaaauCfuUfgcucasusg 1419 CAUGAGCAAGAUUUGCAACUUGC 1509 AD-518811.1 asasacguUfcUfGfGfugucugacuuL96 1330 asAfsgucAfgAfCfaccaGfaAfcguuususc 1420 GAAAACGUUCUGGUGUCUGACUU 1510 AD-520059.1 asascaccUfuUfUfUfcaccuaacuuL96 1331 asAfsguuAfgGfUfgaaaAfaGfguguuscsu 1421 AGAACACCUUUUUCACCUAACUA 1511 AD-519616.1 cscsaccuUfuCfCfCfaguuuuucauL96 1332 asUfsgaaAfaAfCfugggAfaAfgguggsasg 1422 CUCCACCUUUCCCAGUUUUUCAC 1512 AD-518655.1 csasggucCfuCfUfCfagaucuuguuL96 1333 asAfscaaGfaUfCfugagAfgGfaccugscsa 1423 UGCAGGUCCUCUCAGAUCUUGUG 1513 AD-519617.1 csasccuuUfcCfCfAfguuuuucacuL96 1334 asGfsugaAfaAfAfcuggGfaAfaggugsgsa 1424 UCCACCUUUCCCAGUUUUUCACU 1514 AD-519307.1 asasagacAfaAfGfGfuggauacauuL96 1335 asAfsuguAfuCfCfaccuUfuGfucuuuscsa 1425 UGAAAGACAAAGGUGGAUACAUG 1515 AD-520065.3 ususuuucAfcCfUfAfacuaaaauauL96 1336 asUfsauuUfuAfGfuuagGfuGfaaaaasgsg 1426 CCUUUUUCACCUAACUAAAAUAA 1516 AD-519323.1 asusgagcAfaGfAfUfuugcaacuuuL96 1337 asAfsaguUfgCfAfaaucUfuGfcucausgsu 1427 ACAUGAGCAAGAUUUGCAACUUG 1517 AD-519331.1 asusuugcAfaCfUfUfgcuacccauuL96 1338 asAfsuggGfuAfGfcaagUfuGfcaaauscsu 1428 AGAUUUGCAACUUGCUACCCAUU 1518 AD-518922.1 gsascaacGfuAfCfCfcuucauugauL96 1339 asUfscaaUfgAfAfggguAfcGfuugucsasc 1429 GUGACAACGUACCCUUCAUUGAU 1519 AD-519339.1 ususgcuaCfcCfAfUfuaggauaauuL96 1340 asAfsuuaUfcCfUfaaugGfgUfagcaasgsu 1430 ACUUGCUACCCAUUAGGAUAAUG 1520 AD-67552.2 usgsccuuCfgAfGfGfauauuuggauL96 1341 asUfsccaAfaUfAfuccuCfgAfaggcasusa 1431 UAUGCCUUCGAGGAUAUUUGGAU 1521 AD-519756.1 gscsugagUfuGfGfUfuuuaugaaauL96 1342 asUfsuucAfuAfAfaaccAfaCfucagcsusc 1432 GAGCUGAGUUGGUUUUAUGAAAA 1522 AD-519333.1 ususgcaaCfuUfGfCfuacccauuauL96 1343 asUfsaauGfgGfUfagcaAfgUfugcaasasu 1433 AUUUGCAACUUGCUACCCAUUAG 1523 AD-519612.1 csuscuccAfcCfUfUfucccaguuuuL96 1344 asAfsaacUfgGfGfaaagGfuGfgagagscsc 1434 GGCUCUCCACCUUUCCCAGUUUU 1524 AD-519762.1 ususgguuUfuAfUfGfaaaagcuaguL96 1345 asCfsuagCfuUfUfucauAfaAfaccaascsu 1435 AGUUGGUUUUAUGAAAAGCUAGG 1525 AD-75265.3 csasugagCfaAfGfAfuuugcaacuuL96 1346 asAfsguuGfcAfAfaucuUfgCfucaugsusa 1436 UACAUGAGCAAGAUUUGCAACUU 1526 AD-67554.5 uscsugagCfuGfAfGfuugguuuuauL96 1347 asUfsaaaAfcCfAfacucAfgCfucagasgsg 1437 CCUCUGAGCUGAGUUGGUUUUAU 1527 AD-518928.1 gsusacccUfuCfAfUfugaugccaauL96 1348 asUfsuggCfaUfCfaaugAfaGfgguacsgsu 1438 ACGUACCCUUCAUUGAUGCCAAA 1528 AD-519578.1 gsusccagCfcUfGfAfacuucuucuuL96 1349 asAfsgaaGfaAfGfuucaGfgCfuggacscsu 1439 AGGUCCAGCCUGAACUUCUUCUU 1529 AD-75277.3 csusugcuAfcCfCfAfuuaggauaauL96 1350 asUfsuauCfcUfAfauggGfuAfgcaagsusu 1440 AACUUGCUACCCAUUAGGAUAAU 1530 AD-519317.1 usgsgauaCfaUfGfAfgcaagauuuuL96 1351 asAfsaauCfuUfGfcucaUfgUfauccascsc 1441 GGUGGAUACAUGAGCAAGAUUUG 1531 AD-67581.5 cscsuauuAfaUfGfGfucagacuguuL96 1352 asAfscagUfcUfGfaccaUfuAfauaggsgsc 1442 GCCCUAUUAAUGGUCAGACUGUU 1532 AD-519753.1 usgsagcuGfaGfUfUfgguuuuauguL96 1353 asCfsauaAfaAfCfcaacUfcAfgcucasgsa 1443 UCUGAGCUGAGUUGGUUUUAUGA 1533 AD-518701.1 asuscuucCfaUfCfCfauccuucaauL96 1354 asUfsugaAfgGfAfuggaUfgGfaagausgsc 1444 GCAUCUUCCAUCCAUCCUUCAAC 1534 AD-519752.1 csusgagcUfgAfGfUfugguuuuauuL96 1355 asAfsuaaAfaCfCfaacuCfaGfcucagsasg 1445 CUCUGAGCUGAGUUGGUUUUAUG 1535 AD-519328.1 csasagauUfuGfCfAfacuugcuacuL96 1356 asGfsuagCfaAfGfuugcAfaAfucuugscsu 1446 AGCAAGAUUUGCAACUUGCUACC 1536 AD-519322.1 ascsaugaGfcAfAfGfauuugcaacuL96 1357 asGfsuugCfaAfAfucuuGfcUfcaugusasu 1447 AUACAUGAGCAAGAUUUGCAACU 1537 AD-519911.1 csusauuaAfuGfGfUfcagacuguuuL96 1358 asAfsacaGfuCfUfgaccAfuUfaauagsgsg 1448 CCCUAUUAAUGGUCAGACUGUUC 1538 AD-519029.1 asgsggaaCfcUfCfUfaccuucucuuL96 1359 asAfsgagAfaGfGfuagaGfgUfucccusgsu 1449 ACAGGGAACCUCUACCUUCUCUC 1539 AD-519913.1 asusuaauGfgUfCfAfgacuguuccuL96 1360 asGfsgaaCfaGfUfcugaCfcAfuuaausasg 1450 CUAUUAAUGGUCAGACUGUUCCA 1540 AD-518924.1 csasacguAfcCfCfUfucauugauguL96 1361 asCfsaucAfaUfGfaaggGfuAfcguugsusc 1451 GACAACGUACCCUUCAUUGAUGC 1541 AD-519766.1 ususuuauGfaAfAfAfgcuaggaaguL96 1362 asCfsuucCfuAfGfcuuuUfcAfuaaaascsc 1452 GGUUUUAUGAAAAGCUAGGAAGC 1542 AD-519069.1 usasugccUfuCfGfAfggauauuuguL96 1363 asCfsaaaUfaUfCfcucgAfaGfgcauasusc 1453 GAUAUGCCUUCGAGGAUAUUUGG 1543 AD-519614.1 csusccacCfuUfUfCfccaguuuuuuL96 1364 asAfsaaaAfcUfGfggaaAfgGfuggagsasg 1454 CUCUCCACCUUUCCCAGUUUUUC 1544 AD-519618.1 ascscuuuCfcCfAfGfuuuuucacuuL96 1365 asAfsgugAfaAfAfacugGfgAfaaggusgsg 1455 CCACCUUUCCCAGUUUUUCACUA 1545 AD-519326.1 asgscaagAfuUfUfGfcaacuugcuuL96 1366 asAfsgcaAfgUfUfgcaaAfuCfuugcuscsa 1456 UGAGCAAGAUUUGCAACUUGCUA 1546 AD-518920.1 gsusgacaAfcGfUfAfcccuucauuuL96 1367 asAfsaugAfaGfGfguacGfuUfgucacsusc 1457 GAGUGACAACGUACCCUUCAUUG 1547 AD-519760.1 asgsuuggUfuUfUfAfugaaaagcuuL96 1368 asAfsgcuUfuUfCfauaaAfaCfcaacuscsa 1458 UGAGUUGGUUUUAUGAAAAGCUA 1548 AD-518813.1 csgsuucuGfgUfGfUfcugacuuucuL96 1369 asGfsaaaGfuCfAfgacaCfcAfgaacgsusu 1459 AACGUUCUGGUGUCUGACUUUCG 1549 AD-519396.1 uscsugccAfuUfGfCfgauuguccauL96 1370 asUfsggaCfaAfUfcgcaAfuGfgcagasusu 1460 AAUCUGCCAUUGCGAUUGUCCAG 1550 AD-519935.1 csasugagGfuUfCfUfuagaaugacuL96 1371 asGfsucaUfuCfUfaagaAfcCfucaugscsu 1461 AGCAUGAGGUUCUUAGAAUGACA 1551 AD-519758.1 usgsaguuGfgUfUfUfuaugaaaaguL96 1372 asCfsuuuUfcAfUfaaaaCfcAfacucasgsc 1462 GCUGAGUUGGUUUUAUGAAAAGC 1552 AD-518831.1 csgsguccAfaAfGfAfcgaagucguuL96 1373 asAfscgaCfuUfCfgucuUfuGfgaccgsasa 1463 UUCGGUCCAAAGACGAAGUCGUG 1553 AD-518923.1 ascsaacgUfaCfCfCfuucauugauuL96 1374 asAfsucaAfuGfAfagggUfaCfguuguscsa 1464 UGACAACGUACCCUUCAUUGAUG 1554 AD-519755.2 asgscugaGfuUfGfGfuuuuaugaauL96 1375 asUfsucaUfaAfAfaccaAfcUfcagcuscsa 1465 UGAGCUGAGUUGGUUUUAUGAAA 1555 AD-520116.1 usgsugaaUfcAfGfUfgagauguuauL96 1376 asUfsaacAfuCfUfcacuGfaUfucacasusa 1466 UAUGUGAAUCAGUGAGAUGUUAG 1556 AD-519093.1 asusucagGfuUfCfUfuggaagagauL96 1377 asUfscucUfuCfCfaagaAfcCfugaausgsc 1467 GCAUUCAGGUUCUUGGAAGAGAA 1557 AD-67588.2 asascguuCfuGfGfUfgucugacuuuL96 1378 asAfsaguCfaGfAfcaccAfgAfacguususu 1468 AAAACGUUCUGGUGUCUGACUUU 1558 AD-519754.3 gsasgcugAfgUfUfGfguuuuaugauL96 1379 asUfscauAfaAfAfccaaCfuCfagcucsasg 1469 CUGAGCUGAGUUGGUUUUAUGAA 1559 AD-519308.1 asasgacaAfaGfGfUfggauacauguL96 1380 asCfsaugUfaUfCfcaccUfuUfgucuususc 1470 GAAAGACAAAGGUGGAUACAUGA 1560 AD-519759.1 gsasguugGfuUfUfUfaugaaaagcuL96 1381 asGfscuuUfuCfAfuaaaAfcCfaacucsasg 1471 CUGAGUUGGUUUUAUGAAAAGCU 1561 AD-519763.1 usgsguuuUfaUfGfAfaaagcuagguL96 1382 asCfscuaGfcUfUfuucaUfaAfaaccasasc 1472 GUUGGUUUUAUGAAAAGCUAGGA 1562 AD-519327.1 gscsaagaUfuUfGfCfaacuugcuauL96 1383 asUfsagcAfaGfUfugcaAfaUfcuugcsusc 1473 GAGCAAGAUUUGCAACUUGCUAC 1563 AD-519761.1 gsusugguUfuUfAfUfgaaaagcuauL96 1384 asUfsagcUfuUfUfcauaAfaAfccaacsusc 1474 GAGUUGGUUUUAUGAAAAGCUAG 1564 AD-518702.1 uscsuuccAfuCfCfAfuccuucaacuL96 1385 asGfsuugAfaGfGfauggAfuGfgaagasusg 1475 CAUCUUCCAUCCAUCCUUCAACU 1565 AD-519094.1 ususcaggUfuCfUfUfggaagagaauL96 1386 asUfsucuCfuUfCfcaagAfaCfcugaasusg 1476 CAUUCAGGUUCUUGGAAGAGAAG 1566 AD-518832.1 gsgsuccaAfaGfAfCfgaagucguguL96 1387 asCfsacgAfcUfUfcgucUfuUfggaccsgsa 1477 UCGGUCCAAAGACGAAGUCGUGG 1567 AD-519767.1 ususuaugAfaAfAfGfcuaggaagcuL96 1388 asGfscuuCfcUfAfgcuuUfuCfauaaasasc 1478 GUUUUAUGAAAAGCUAGGAAGCA 1568 AD-518917.1 usgsagugAfcAfAfCfguacccuucuL96 1389 asGfsaagGfgUfAfcguuGfuCfacucascsu 1479 AGUGAGUGACAACGUACCCUUCA 1569 AD-518988.1 csusucauGfuGfGfAfcaucaccaauL96 1390 asUfsuggUfgAfUfguccAfcAfugaagsasa 1480 UUCUUCAUGUGGACAUCACCAAG 1570 AD-519305.1 usgsaaagAfcAfAfAfgguggauacuL96 1391 asGfsuauCfcAfCfcuuuGfuCfuuucasusu 1481 AAUGAAAGACAAAGGUGGAUACA 1571 AD-519613.1 uscsuccaCfcUfUfUfcccaguuuuuL96 1392 asAfsaaaCfuGfGfgaaaGfgUfggagasgsc 1482 GCUCUCCACCUUUCCCAGUUUUU 1572 AD-519121.1 cscsugaaGfuCfAfUfccucagaaguL96 1393 asCfsuucUfgAfGfgaugAfcUfucaggscsc 1483 GGCCUGAAGUCAUCCUCAGAAGG 1573 AD-519095.1 uscsagguUfcUfUfGfgaagagaaguL96 1394 asCfsuucUfcUfUfccaaGfaAfccugasasu 1484 AUUCAGGUUCUUGGAAGAGAAGG 1574 AD-518982.1 asascuuuCfuUfCfAfuguggacauuL96 1395 asAfsuguCfcAfCfaugaAfgAfaaguuscsg 1485 CGAACUUUCUUCAUGUGGACAUC 1575 AD-519096.1 csasgguuCfuUfGfGfaagagaagguL96 1396 asCfscuuCfuCfUfuccaAfgAfaccugsasa 1486 UUCAGGUUCUUGGAAGAGAAGGG 1576 AD-518986.1 ususcuucAfuGfUfGfgacaucaccuL96 1397 asGfsgugAfuGfUfccacAfuGfaagaasasg 1487 CUUUCUUCAUGUGGACAUCACCA 1577 AD-518921.1 usgsacaaCfgUfAfCfccuucauuguL96 1398 asCfsaauGfaAfGfgguaCfgUfugucascsu 1488 AGUGACAACGUACCCUUCAUUGA 1578 AD-518985.1 ususucuuCfaUfGfUfggacaucacuL96 1399 asGfsugaUfgUfCfcacaUfgAfagaaasgsu 1489 ACUUUCUUCAUGUGGACAUCACC 1579 AD-518984.1 csusuucuUfcAfUfGfuggacaucauL96 1400 asUfsgauGfuCfCfacauGfaAfgaaagsusu 1490 AACUUUCUUCAUGUGGACAUCAC 1580 AD-519325.1 gsasgcaaGfaUfUfUfgcaacuugcuL96 1401 asGfscaaGfuUfGfcaaaUfcUfugcucsasu 1491 AUGAGCAAGAUUUGCAACUUGCU 1581 AD-518812.1 ascsguucUfgGfUfGfucugacuuuuL96 1402 asAfsaagUfcAfGfacacCfaGfaacgususu 1492 AAACGUUCUGGUGUCUGACUUUC 1582 AD-518987.1 uscsuucaUfgUfGfGfacaucaccauL96 1403 asUfsgguGfaUfGfuccaCfaUfgaagasasa 1493 UUUCUUCAUGUGGACAUCACCAA 1583 AD-518958.1 csasucugCfcCfUfAfaagucaaguuL96 1404 asAfscuuGfaCfUfuuagGfgCfagaugsusc 1494 GACAUCUGCCCUAAAGUCAAGUC 1584 AD-67572.2 ususcuuuCfaGfAfGfgugcuaaaguL96 1405 asCfsuuuAfgCfAfccucUfgAfaagaasusc 1495 GAUUCUUUCAGAGGUGCUAAAGU 1585 AD-518980.1 csgsaacuUfuCfUfUfcauguggacuL96 1406 asGfsuccAfcAfUfgaagAfaAfguucgsusg 1496 CACGAACUUUCUUCAUGUGGACA 1586 AD-519070.1 asusgccuUfcGfAfGfgauauuugguL96 1407 asCfscaaAfuAfUfccucGfaAfggcausasu 1497 AUAUGCCUUCGAGGAUAUUUGGA 1587 AD-518925.1 asascguaCfcCfUfUfcauugaugcuL96 1408 asGfscauCfaAfUfgaagGfgUfacguusgsu 1498 ACAACGUACCCUUCAUUGAUGCC 1588 AD-518981.1 gsasacuuUfcUfUfCfauguggacauL96 1409 asUfsgucCfaCfAfugaaGfaAfaguucsgsu 1499 ACGAACUUUCUUCAUGUGGACAU 1589

TABLE 10 Unmodified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ ID ID Duplex Name Sense Sequences NO: Range Antisense Sequences NO: Range AD-521086.1 ACAGGGAACCUCUACCUUU 1590 819-837 AAAGGUAGAGGUUCCCUGU 1626 AD-805632.1 ACAGGGAACCUCUACCUUU 1591 AAAGGUAGAGGUUCCCUGU 1627 819-837 AD-805644.1 ACAGGGAACCUCUACCUUU 1592 AAAGGUAGAGGUUCCCUGU 1628 AD-521465.1 CCAUUGCGAUUGUCCAGAU 1593 1267-1285 AUCUGGACAAUCGCAAUGG 1629 AD-805631.1 CCAUUGCGAUUGUCCAGAU 1594 AUCUGGACAAUCGCAAUGG 1630 1267-1285 AD-805643.1 CCAUUGCGAUUGUCCAGAU 1595 AUCUGGACAAUCGCAAUGG 1631 AD-520902.1 CGAAGUCGUGGAUGCCUUU 1596 581-599 AAAGGCAUCCACGACUUCG 1632 581-599 AD-805634.1 CGAAGUCGUGGAUGCCUUU 1597 AAAGGCAUCCACGACUUCG 1633 AD-805646.1 CGAAGUCGUGGAUGCCUUU 1598 AAAGGCAUCCACGACUUCG 1634 AD-522140.1 CUUUUUCACCUAACUAAAU 1599 2182-2200 AUUUAGUUAGGUGAAAAAG 1635 AD-805635.1 CUUUUUCACCUAACUAAAU 1600 AUUUAGUUAGGUGAAAAAG 1636 2182-2200 AD-805647.1 CUUUUUCACCUAACUAAAU 1601 AUUUAGTUAGGUGAAAAAG 1637 AD-520903.1 GAAGUCGUGGAUGCCUUGU 1602 582-600 ACAAGGCAUCCACGACUUC 1638 AD-805628.1 GAAGUCGUGGAUGCCUUGU 1603 ACAAGGCAUCCACGACUUC 1639 582-600 AD-805640.1 GAAGUCGUGGAUGCCUUGU 1604 ACAAGGCAUCCACGACUUC 1640 AD-520973.1 GAGUGAGUGACAACGUACU 1605 676-694 AGUACGUUGUCACUCACUC 1641 AD-805625.1 GAGUGAGUGACAACGUACU 1606 AGUACGUUGUCACUCACUC 1642 676-694 AD-805637.1 GAGUGAGUGACAACGUACU 1607 AGUACGTUGUCACUCACUC 1643 AD-521129.1 GAUAUGCCUUCGAGGAUAU 1608 881-899 AUAUCCUCGAAGGCAUAUC 1644 AD-805630.1 GAUAUGCCUUCGAGGAUAU 1609 AUAUCCUCGAAGGCAUAUC 1645 881-899 AD-805642.1 GAUAUGCCUUCGAGGAUAU 1610 AUAUCCTCGAAGGCAUAUC 1646 AD-521124.1 GGAGAGAUAUGCCUUCGAU 1611 876-894 AUCGAAGGCAUAUCUCUCC 1647 AD-805626.1 GGAGAGAUAUGCCUUCGAU 1612 AUCGAAGGCAUAUCUCUCC 1648 876-894 AD-805638.1 GGAGAGAUAUGCCUUCGAU 1613 AUCGAAGGCAUAUCUCUCC 1649 AD-521420.1 GGAUAAUGUCUUAUGUAAU 1614 1219-1237 AUUACAUAAGACAUUAUCC 1650 AD-805624.1 GGAUAAUGUCUUAUGUAAU 1615 AUUACAUAAGACAUUAUCC 1651 1219-1237 AD-805636.1 GGAUAAUGUCUUAUGUAAU 1616 AUUACATAAGACAUUAUCC 1652 AD-521840.1 GGUUUUAUGAAAAGCUAGU 1617 1753-1771 ACUAGCUUUUCAUAAAACC 1653 AD-805629.1 GGUUUUAUGAAAAGCUAGU 1618 ACUAGCUUUUCAUAAAACC 1654 1753-1771 AD-805641.1 GGUUUUAUGAAAAGCUAGU 1619 ACUAGCTUUUCAUAAAACC 1655 AD-521409.1 UGCUACCCAUUAGGAUAAU 1620 1207-1225 AUUAUCCUAAUGGGUAGCA 1656 AD-805633.1 UGCUACCCAUUAGGAUAAU 1621 AUUAUCCUAAUGGGUAGCA 1657 1207-1225 AD-805645.1 UGCUACCCAUUAGGAUAAU 1622 AUUAUCCUAAUGGGUAGCA 1658 AD-521486.1 UGGUGACAUGGCUUCCAGU 1623 1288-1306 ACUGGAAGCCAUGUCACCA 1659 AD-805627.1 UGGUGACAUGGCUUCCAGU 1624 ACUGGAAGCCAUGUCACCA 1660 1288-1306 AD-805639.1 UGGUGACAUGGCUUCCAGU 1625 ACUGGAAGCCAUGUCACCA 1661

TABLE 11 Modified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ SEQ ID ID mRNA ID Duplex Name Sense sequence NO: Antisense Sequence NO: Target Sequence NO: AD-521086.1 ACAGGGAACCUCUACCUUUdTdT 1662 AAAGGUAGAGGUUCCCUGUdTdT 1698 ACAGGGAACCUCUACCUUC 1734 AD-805632.1 ascsagggAfaCfCfUfcuaccuuuL96 1663 asAfsaggUfagagguuCfcCfugu 1699 AD-805644.1 ascsagggAfaCfCfUfcuaccuuuL96 1664 asAfsaggUf(Agn)gagguuCfcCfugu 1700 AD-521465.1 CCAUUGCGAUUGUCCAGAUdTdT 1665 AUCUGGACAAUCGCAAUGGdTdT 1701 CCAUUGCGAUUGUCCAGAG 1735 AD-805631.1 cscsauugCfgAfUfUfguccagauL96 1666 asUfscugGfacaaucgCfaAfugg 1702 AD-805643.1 cscsauugCfgAfUfUfguccagauL96 1667 asUfscugGf(Agn)caaucgCfaAfugg 1703 AD-520902.1 CGAAGUCGUGGAUGCCUUUdTdT 1668 AAAGGCAUCCACGACUUCGdTdT 1704 CGAAGUCGUGGAUGCCUUG 1736 AD-805634.1 csgsaaguCfgUfGfGfaugccuuuL96 1669 asAfsaggCfauccacgAfcUfucg 1705 AD-805646.1 csgsaaguCfgUfGfGfaugccuuuL96 1670 asAfsaggCf(Agn)uccacgAfcUfucg 1706 AD-522140.1 CUUUUUCACCUAACUAAAUdTdT 1671 AUUUAGUUAGGUGAAAAAGdTdT 1707 CUUUUUCACCUAACUAAAA 1737 AD-805635.1 csusuuuuCfaCfCfUfaacuaaauL96 1672 asUfsuuaGfuuaggugAfaAfaag 1708 AD-805647.1 csusuuuuCfaCfCfUfaacuaaauL96 1673 asUfsuuaGf(Tgn)uaggugAfaAfaag 1709 AD-520903.1 GAAGUCGUGGAUGCCUUGUdTdT 1674 ACAAGGCAUCCACGACUUCdTdT 1710 GAAGUCGUGGAUGCCUUGG 1738 AD-805628.1 gsasagucGfuGfGfAfugccuuguL96 1675 asCfsaagGfcauccacGfaCfuuc 1711 AD-805640.1 gsasagucGfuGfGfAfugccuuguL96 1676 asCfsaagGf(Cgn)auccacGfaCfuuc 1712 AD-520973.1 GAGUGAGUGACAACGUACUdTdT 1677 AGUACGUUGUCACUCACUCdTdT 1713 GAGUGAGUGACAACGUACC 1739 AD-805625.1 gsasgugaGfuGfAfCfaacguacuL96 1678 asGfsuacGfuugucacUfcAfcuc 1714 AD-805637.1 gsasgugaGfuGfAfCfaacguacuL96 1679 asGfsuacGf(Tgn)ugucacUfcAfcuc 1715 AD-521129.1 GAUAUGCCUUCGAGGAUAUdTdT 1680 AUAUCCUCGAAGGCAUAUCdTdT 1716 GAUAUGCCUUCGAGGAUAU 1740 AD-805630.1 gsasuaugCfcUfUfCfgaggauauL96 1681 asUfsaucCfucgaaggCfaUfauc 1717 AD-805642.1 gsasuaugCfcUfUfCfgaggauauL96 1682 asUfsaucCf(Tgn)cgaaggCfaUfauc 1718 AD-521124.1 GGAGAGAUAUGCCUUCGAUdTdT 1683 AUCGAAGGCAUAUCUCUCCdTdT 1719 GGAGAGAUAUGCCUUCGAG 1741 AD-805626.1 gsgsagagAfuAfUfGfccuucgauL96 1684 asUfscgaAfggcauauCfuCfucc 1720 AD-805638.1 gsgsagagAfuAfUfGfccuucgauL96 1685 asUfscgaAf(Ggn)gcauauCfuCfucc 1721 AD-521420.1 GGAUAAUGUCUUAUGUAAUdTdT 1686 AUUACAUAAGACAUUAUCCdTdT 1722 GGAUAAUGUCUUAUGUAAU 1742 AD-805624.1 gsgsauaaUfgUfCfUfuauguaauL96 1687 asUfsuacAfuaagacaUfuAfucc 1723 AD-805636.1 gsgsauaaUfgUfCfUfuauguaauL96 1688 asUfsuacAf(Tgn)aagacaUfuAfucc 1724 AD-521840.1 GGUUUUAUGAAAAGCUAGUdTdT 1689 ACUAGCUUUUCAUAAAACCdTdT 1725 GGUUUUAUGAAAAGCUAGG 1743 AD-805629.1 gsgsuuuuAfuGfAfAfaagcuaguL96 1690 asCfsuagCfuuuucauAfaAfacc 1726 AD-805641.1 gsgsuuuuAfuGfAfAfaagcuaguL96 1691 asCfsuagCf(Tgn)uuucauAfaAfacc 1727 AD-521409.1 UGCUACCCAUUAGGAUAAUdTdT 1692 AUUAUCCUAAUGGGUAGCAdTdT 1728 UGCUACCCAUUAGGAUAAU 1744 AD-805633.1 usgscuacCfcAfUfUfaggauaauL96 1693 asUfsuauCfcuaauggGfuAfgca 1729 AD-805645.1 usgscuacCfcAfUfUfaggauaauL96 1694 asUfsuauCf(Cgn)uaauggGfuAfgca 1730 AD-521486.1 UGGUGACAUGGCUUCCAGUdTdT 1695 ACUGGAAGCCAUGUCACCAdTdT 1731 UGGUGACAUGGCUUCCAGA 1745 AD-805627.1 usgsgugaCfaUfGfGfcuuccaguL96 1696 asCfsuggAfagccaugUfcAfcca 1732 AD-805639.1 usgsgugaCfaUfGfGfcuuccaguL96 1697 asCfsuggAf(Agn)gccaugUfcAfcca 1733

TABLE 12 PNPLA3 Single Dose Screen in Cos-7 Cells (Human Dual-Luciferase psiCHECK2 vector) 50 nM 10 nM 1 nM 0.1 nM % of % of % of % of Message ST Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEV AD-517197.1  17.0  7.0 15.4 3.4  41.0 13.9 77.8 16.2 AD-516851.1  22.8  5.7 38.8 15  63.1 16.0 94.6 6.8 AD-516748.1  26.8 15.5 61.7 10.7  59.0 38.3 95.4 27.2 AD-517234.1  31.1  7.3 39.2 3  69.0 10.0 98.7 18.5 AD-517354.1  32.0  7.4 42 9.8 102.2 29.0 98.4 15.4 AD-517257.1  33.3  7.5 41.8 12.3  63.9 16.7 96.8 12.4 AD-516739.1  33.3 10.2 50.7 9  85.6 21.7 110.6 13.9 AD-517258.1  35.2 14.3 27.9 6.3  66.6 20.7 94.2 10.4 AD-516629.1  36.5  8.3 47.6 13.2  94.6  9.0 126.1 22 AD-516972.1  37.0 12.8 51.5 1.6 113.3 23.9 135.9 22.9 AD-517623.1  37.5 12.5 55.9 7  76.6 25.6 96.9 12.7 AD-516733.1  37.7 11.4 47.4 14.4  87.2  7.0 96.5 13 AD-517985.1  40.4  9.2 51.2 10.3  78.6 13.9 98.4 14.4 AD-516827.1  40.7 24.3 60.1 27.3  81.5 45.6 90.2 9.2 AD-516917.1  42.1 13.8 35.1 8.3  69.4 15.4 99.6 22.2 AD-516973.1  43.0  2.8 70.8 9.6 100.4 17.1 114.4 24.2 AD-516978.1  44.3 18.5 46.1 18.4  76.2  6.9 99 13 AD-517310.1  45.5 15.4 61.1 13  89.7 10.9 128.5 14.6 AD-516828.1  46.2 12.1 55.8 12.6  95.9 15.9 120.4 30.5 AD-517249.1  46.6 14.7 37.6 9  84.3  9.6 115.2 19.5 AD-517196.1  47.5 14.0 55.2 7.3  82.3 13.5 116.3 22.9 AD-517322.1  47.7 13.6 89.7 23.1 101.5 21.4 95.9 5.6 AD-517319.1  48.4 29.0 70 24.7  90.6 15.9 125.1 5.6 AD-516822.1  49.1 15.4 44.3 12.6  78.6 12.3 100 11.9 AD-516826.1  50.6 17.1 73.2 20.1  90.1  4.2 115.9 13.8 AD-516824.1  51.5  7.7 67.1 6.6  99.4 24.5 112.9 11.6 AD-517517.1  51.5 11.0 80.6 16  79.8 17.5 113.5 7.3 AD-517758.1  52.1 17.6 69.2 3.1 109.3  5.7 106.9 6.5 AD-516940.1  52.6 27.2 82.7 27.6  79.6 15.9 88.6 15.7 AD-517318.1  54.1 23.3 54.6 14.1 105.9 13.4 102.4 22.4 AD-517321.1  54.3 22.5 63.6 32.3  92.5  6.7 98.5 11 AD-516747.1  54.3 22.4 62.2 12.2  79.6 16.4 126.5 11.9 AD-516737.1  54.4  4.2 74.3 19.7  76.8  6.9 93.4 13.4 AD-516742.1  54.6 12.8 70.8 10 115.8 28.5 104 19.3 AD-516977.1  55.6 20.2 63.4 15.1  83.6 11.5 97.4 22.3 AD-516823.1  56.5 24.3 65.8 16.6  85.9 18.5 90.8 8.6 AD-516871.1  56.9 17.2 62.6 9.3 101.4 18.0 113.7 18.5 AD-516771.1  57.1  8.8 66.2 10.6  82.1  9.6 100.8 19.5 AD-517757.1  57.5  9.1 72.6 9 100.2  5.8 97.6 7.1 AD-516745.1  58.3 31.2 60.6 12.4  91.2 11.5 133 33.3 AD-517830.1  60.1 18.0 75.2 11.8  85.7  5.7 92 13 AD-516970.1  60.7  9.9 81.7 18.6 104.3  5.3 100.2 4.8 AD-517768.1  61.9  5.8 89.5 20 109.8 32.6 113.8 31.4 AD-517259.1  62.4 29.7 73.9 13.4  94.2 26.1 95.2 27.4 AD-516979.1  65.1 18.5 70.3 34.6  92.6 17.3 101.3 15.6 AD-516971.1  65.2 39.8 81.4 26.5 107.8  8.6 126 56.3 AD-517838.1  66.5  7.3 57 12  96.9 21.2 113.8 8.1 AD-516743.1  66.5 10.8 76.9 20.6  74.9  9.3 110.1 14.2 AD-516980.1  66.8 32.1 73.1 12.6 110.6  4.4 111.4 5.9 AD-517771.1  67.7 18.0 77.6 20.1  91.0 21.5 99 6 AD-516772.1  69.8  9.1 74 10.6 111.8 30.0 103.9 17.8 AD-517836.1  69.9 40.1 69 20.6  97.2 32.9 115.4 22.5 AD-516741.1  69.9 24.6 96.6 22.8 100.4 13.9 125.8 20.3 AD-517353.1  70.7 27.0 93.2 10.7  98.8 21.7 116 24.8 AD-517979.1  72.5 10.1 88 26.7 113.4  6.8 139 10.6 AD-516937.1  73.5 14.3 71.5 12.7  92.2 18.2 115.9 12.9 AD-516976.1  73.5 38.4 59.6 12.7  73.5  4.8 90 12.7 AD-516872.1  73.6 37.1 56.2 10.9  87.7 20.6 101.1 19 AD-517256.1  73.9 24.1 98.1 8.7 105.0 10.9 99.6 6.7 AD-516825.1  74.1 11.0 84.8 25.3 100.9 25.6 102 13.4 AD-516735.1  74.5  7.3 98.9 6.8  95.4 12.5 98.8 10.7 AD-516588.1  75.2 28.1 88.5 13 112.2 18.2 122.4 25.5 AD-516738.1  75.6 15.0 85.1 17  89.8 27.8 131.4 32.6 AD-517314.1  76.8 24.6 69.7 18  81.0 16.6 93.2 10.4 AD-517805.1  76.9  9.4 70.6 9 112.4 26.3 96.4 17.7 AD-517685.1  78.3 22.9 64.6 6.6  98.6 12.9 108.2 19.8 AD-517831.1  78.8 13.5 91.4 18.1 118.3 21.8 131.6 23.2 AD-516830.1  78.9  9.0 81.5 22.5  91.1 17.5 108 32.5 AD-517837.1  79.5 53.8 68.2 22.7  97.8 33.2 96.8 12.9 AD-517633.1  81.1 21.5 89.9 10.7  91.2 17.6 93.9 9.4 AD-516855.1  81.3 21.1 74.4 11.6  93.0 19.8 111 9.5 AD-516688.1  83.0 33.6 69.9 9.3  80.1 10.5 106.2 13.8 AD-516630.1  86.4 33.1 82 22.2  69.8 23.2 92 15.4 AD-516835.1  86.8 17.0 70.5 13.3 103.1 17.2 101.4 8.2 AD-516832.1  87.4 14.5 100 20.5 108.9 11.7 127.3 24.9 AD-517834.1  88.9 21.8 73.9 9.1  90.4 13.8 79.3 8.5 AD-516734.1  89.7 19.7 88.3 12.6  98.6  5.2 103.4 23.2 AD-517228.1  89.7 14.3 83.5 17.5 101.4 19.9 118.8 17 AD-516736.1  93.2 12.9 90.2 19.6 115.9 14.4 106.7 5.4 AD-517646.1  93.8 38.8 71.9 18.9 104.5 24.7 100.1 10.7 AD-517744.1  95.6 10.2 80.5 10.4  85.3 14.9 99.9 16 AD-517509.1  96.7 20.8 108 11.2  95.6 15.9 124.2 20.1 AD-517746.1  97.6 13.6 79.3 19.8  91.4 16.7 94 17.1 AD-516752.1  99.9 29.7 94 25.5 105.4 18.9 117.7 13.1 AD-516746.1 100.3 19.5 80.5 14.1 100.5 22.4 116.2 6.8 AD-517227.1 102.2 25.7 79.3 10  90.6 22.5 115.3 21.8 AD-516751.1  54.2 31.7 51.8 11.2  91.2  8.5 125.7 19.2 AD-517042.1 102.4 31.9 64.7 8.2  90.2  6.3 113.1 19.6 AD-517571.1  85.2 54.8 88.7 17.9  87.5  8.1 93.3 6.4

TABLE 13 PNPLA3 Single Dose Screen in Cos-7 Cells (Human Dual-Luciferase psiCHECK2 vector) 50 nM 10 nM 1 nM 0.1 nM % of % of % of % of Message ST Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEV AD-67605.6  7.2  7.9  34.8 14.9  66.5 24.1  93.0 19.7 AD-520101.1 13.0 10.3  39.4 18.8  77.1 12.9  74.1 23.0 AD-520098.1 18.5  5.3  65.4 41.4  68.0 30.3 114.7 42.8 AD-67575.6 19.3  6.6  33.8 23.9  89.4 29.7 105.2 38.7 AD-520467.1 20.5 13.6  46.8 19.1  72.7 12.1 133.1 81.1 AD-520064.1 21.2  4.3  29.5  9.0  58.8  6.1 110.0 23.0 AD-520099.1 21.2 13.1  64.7  9.7  61.3 22.5 100.6 38.3 AD-520466.1 22.2 10.3  52.4  8.8  77.4 24.1 141.2 54.5 AD-519351.1 22.9 18.1  28.3  6.7  50.3 18.5  91.2 43.4 AD-520065.1 23.4 21.2  49.6  5.2  55.3 24.7  90.4 11.3 AD-520069.1 23.8 18.5  65.1  5.3  98.6 21.1 134.8 27.2 AD-519828.1 23.9 23.3  41.5 24.7 100.1 24.7  99.6 21.4 AD-520035.1 25.8 18.4  57.3 24.0 106.9 34.4 100.2 27.7 AD-520067.1 28.5 24.7  38.1 17.6  63.1 20.7  96.6 20.9 AD-75289.2 28.5 16.3  44.1 20.5  81.8 18.6  82.1  7.3 AD-520125.1 29.4 15.6  57.1 10.4  76.6 14.5 155.3 45.2 AD-520018.1 29.9  6.3  32.0 16.0  66.1 19.8 101.4 39.1 AD-520062.1 31.4  9.1  53.5 36.7  66.8 23.2  74.9 14.7 AD-519754.1 32.6  7.3  86.3 24.9  98.3 27.4  99.9 16.2 AD-520097.1 35.2 16.8  38.7  9.5  95.2 34.8 123.6 33.6 AD-520352.1 37.0 14.5  63.4 19.8  77.9 26.9 105.2 25.3 AD-519755.1 38.8 21.4  48.7 19.1 101.4 25.3 130.8 48.1 AD-520063.1 40.2 12.8  58.5 39.1  56.3 19.0 107.1 24.9 AD-520066.1 43.2 13.7  40.5 11.4  58.2 18.3 115.4 46.6 AD-520068.1 46.8 22.3  53.0 43.8  68.9 41.3  90.5 40.0 AD-520465.1 66.5 14.2 101.1 35.3  71.4 32.2  88.7 12.6 AD-519592.1 67.2 13.0  99.4 40.7  69.9 16.9  87.1 23.7 AD-519591.1 75.9 33.3  79.8 23.0  95.9 36.6  89.6 33.9

TABLE 14 PNPLA3 Single Dose Screen in Hep3B Cells Avg % of Message Dose Duplex Remaining SD (nM) AD-519351.1 18.91743 1.592235 50 AD-519591.1 30.15831 4.008696 50 AD-519592.1 49.60929 3.258342 50 AD-519754.1 29.95629 3.489596 50 AD-519755.1 32.7814 3.836985 50 AD-519828.1 25.91768 2.010603 50 AD-520018.1 17.85763 0.704719 50 AD-520035.1 13.40537 0.824367 50 AD-520062.1 16.07361 2.76391 50 AD-520063.1 42.26994 7.472034 50 AD-520064.1 32.07188 6.038421 50 AD-520065.1 41.04576 7.358829 50 AD-520066.1 49.84015 4.399835 50 AD-520067.1 32.29406 2.439182 50 AD-520068.1 34.46787 3.933021 50 AD-520069.1 50.74699 7.696823 50 AD-520097.1 37.05357 3.157405 50 AD-520098.1 43.45243 7.096347 50 AD-520099.1 34.3067 6.026941 50 AD-520101.1 34.10031 2.946968 50 AD-520125.1 37.9648 5.052388 50 AD-520352.1 55.35492 5.581261 50 AD-520465.1 116.2403 11.93776 50 AD-520466.1 50.49998 5.242516 50 AD-520467.1 57.64928 4.998672 50 AD-67575.6 32.8979 5.107761 50 AD-67605.6 30.30416 5.607134 50 AD-75289.2 37.28977 7.002537 50 AD-519351.1 28.011 2.78131 10 AD-519591.1 48.39234 9.625783 10 AD-519592.1 67.92064 14.89226 10 AD-519754.1 51.1618 7.649766 10 AD-519755.1 41.00894 4.838157 10 AD-519828.1 39.08326 3.07126 10 AD-520018.1 14.21871 7.205605 10 AD-520035.1 17.60407 3.934564 10 AD-520062.1 25.84533 3.490245 10 AD-520063.1 42.59087 3.989656 10 AD-520064.1 32.13541 3.984171 10 AD-520065.1 34.56078 6.902658 10 AD-520066.1 43.89564 5.960707 10 AD-520067.1 30.99356 5.829848 10 AD-520068.1 44.00221 5.902595 10 AD-520069.1 51.01245 6.208729 10 AD-520097.1 44.51291 6.137725 10 AD-520098.1 45.04234 3.412759 10 AD-520099.1 39.05479 4.499439 10 AD-520101.1 38.56051 7.725309 10 AD-520125.1 54.31024 10.76057 10 AD-520352.1 51.88458 9.725295 10 AD-520465.1 93.6487 8.10188 10 AD-520466.1 60.38535 6.422916 10 AD-520467.1 50.73771 8.167229 10 AD-67575.6 42.36839 6.376496 10 AD-67605.6 32.93356 4.534181 10 AD-75289.2 35.10034 4.252315 10 AD-519351.1 38.91029 6.73621 1 AD-519591.1 72.03301 8.409019 1 AD-519592.1 91.99647 6.557726 1 AD-519754.1 71.39233 10.16091 1 AD-519755.1 48.64478 8.703204 1 AD-519828.1 56.57794 8.681994 1 AD-520018.1 43.24255 12.35058 1 AD-520035.1 41.61261 9.606674 1 AD-520062.1 41.56298 7.015498 1 AD-520063.1 50.9099 8.41448 1 AD-520064.1 49.60309 8.547469 1 AD-520065.1 51.99888 13.42055 1 AD-520066.1 58.14913 6.189735 1 AD-520067.1 47.67786 4.442494 1 AD-520068.1 68.81371 17.00633 1 AD-520069.1 58.92955 8.034131 1 AD-520097.1 53.79314 4.302369 1 AD-520098.1 62.39227 4.285866 1 AD-520099.1 74.81884 4.665135 1 AD-520101.1 59.49561 9.59846 1 AD-520125.1 60.19627 4.672244 1 AD-520352.1 79.06617 11.33413 1 AD-520465.1 103.3435 26.3229 1 AD-520466.1 86.98648 21.00735 1 AD-520467.1 81.16465 23.24505 1 AD-67575.6 59.97269 7.272857 1 AD-67605.6 52.64283 5.165939 1 AD-75289.2 73.81446 13.1944 1 AD-519351.1 58.00814 5.565421 0.1 AD-519591.1 88.58883 14.40971 0.1 AD-519592.1 100.9556 9.227538 0.1 AD-519754.1 93.4398 15.32662 0.1 AD-519755.1 79.44622 3.664871 0.1 AD-519828.1 93.29187 9.598122 0.1 AD-520018.1 67.61844 8.626892 0.1 AD-520035.1 60.53901 5.277965 0.1 AD-520062.1 72.05043 8.551113 0.1 AD-520063.1 79.2352 11.96642 0.1 AD-520064.1 64.04505 10.59475 0.1 AD-520065.1 74.87369 15.32438 0.1 AD-520066.1 77.48361 11.28607 0.1 AD-520067.1 79.16226 8.228879 0.1 AD-520068.1 98.42636 29.19873 0.1 AD-520069.1 81.92734 10.02953 0.1 AD-520097.1 78.17625 13.3457 0.1 AD-520098.1 81.87427 12.05935 0.1 AD-520099.1 87.99157 22.24171 0.1 AD-520101.1 90.27513 9.407839 0.1 AD-520125.1 91.10768 17.63111 0.1 AD-520352.1 99.6731 4.089456 0.1 AD-520465.1 89.46856 1.073905 0.1 AD-520466.1 94.49258 4.863811 0.1 AD-520467.1 91.43145 12.05283 0.1 AD-67575.6 86.88788 17.45404 0.1 AD-67605.6 89.55684 14.21808 0.1 AD-75289.2 95.69357 10.92256 0.1

TABLE 15 PNPLA3 Single Dose Screen in Cos-7 Cells (Human Dual-Luciferase psiCHECK2 vector) 50 nM % of Message ST DuplexID Remaining DEV AD-521420.1 12.6 5.2 AD-520973.1 13.0 9.6 AD-521124.1 15.6 12.7 AD-521486.1 17.6 4.6 AD-520903.1 17.6 14.3 AD-520972.1 17.6 7.4 AD-521421.1 17.7 6.7 AD-521840.1 17.8 3.3 AD-521003.1 18.1 6.4 AD-521129.1 18.9 5.7 AD-521465.1 19.2 10.1 AD-521086.1 19.5 7.9 AD-521409.1 20.5 12.1 AD-522178.1 20.6 11.7 AD-520974.1 22.0 8.0 AD-520902.1 24.1 9.4 AD-522140.1 24.3 4.9 AD-521410.1 25.6 7.2 AD-522548.1 26.1 3.8 AD-521002.1 26.1 9.0 AD-522176.1 27.2 5.2 AD-520926.1 28.1 12.4 AD-521895.1 28.3 7.9 AD-521499.1 29.1 4.9 AD-521466.1 29.2 6.9 AD-521140.1 29.5 9.4 AD-520892.1 30.1 11.7 AD-520976.1 30.5 13.4 AD-521457.1 30.6 11.5 AD-521127.1 30.7 7.8 AD-522145.1 31.2 10.2 AD-520984.1 31.2 9.1 AD-521997.1 32.2 15.4 AD-522174.1 32.8 9.7 AD-522545.1 33.6 7.1 AD-521979.1 33.8 6.7 AD-520891.1 33.9 14.8 AD-521833.1 34.3 9.6 AD-521461.1 35.0 22.6 AD-521386.1 35.0 15.0 AD-521123.1 35.1 23.9 AD-520899.1 35.9 14.2 AD-521089.1 36.9 26.5 AD-521407.1 37.3 15.3 AD-520898.1 38.6 18.6 AD-521378.1 38.7 18.6 AD-521500.1 38.8 17.7 AD-521798.1 39.4 14.3 AD-521902.1 39.6 10.0 AD-521896.1 39.8 18.4 AD-521989.1 39.8 15.6 AD-520896.1 40.7 13.9 AD-69024.2 41.1 8.3 AD-522146.1 41.6 9.7 AD-522432.1 41.9 18.9 AD-521020.1 42.2 11.6 AD-521668.1 42.3 8.2 AD-522097.1 43.0 12.5 AD-520999.1 43.9 18.0 AD-521832.1 44.4 10.4 AD-520894.1 44.7 10.5 AD-522144.1 45.0 22.2 AD-521408.1 45.2 24.2 AD-521128.1 45.7 9.9 AD-521980.1 45.8 5.0 AD-521406.1 46.5 17.9 AD-521131.1 46.8 21.4 AD-521909.1 47.1 28.4 AD-521954.1 47.4 7.8 AD-520872.1 47.7 19.8 AD-522142.1 48.2 22.3 AD-520994.1 48.8 6.2 AD-520886.1 50.0 20.3 AD-521987.1 50.5 17.9 AD-521988.1 50.8 6.9 AD-520785.1 51.2 11.0 AD-521919.1 51.4 18.6 AD-521121.1 51.7 15.0 AD-522202.1 53.1 17.3 AD-521130.1 54.5 17.6 AD-521908.1 55.1 33.8 AD-522173.1 55.1 15.5 AD-521785.1 56.1 13.2 AD-520890.1 56.3 19.6 AD-520978.1 56.5 26.3 AD-521744.1 57.5 6.9 AD-521021.1 58.1 4.4 AD-521197.1 59.2 18.9 AD-521379.1 59.7 42.4 AD-520925.1 60.6 12.4 AD-520888.1 60.8 24.3 AD-520975.1 66.0 16.0 AD-521666.1 66.3 30.6 AD-521922.1 66.5 19.7 AD-521986.1 67.3 21.9 AD-521915.1 67.6 21.7 AD-520897.1 69.4 28.1 AD-520889.1 73.3 27.6 AD-520885.1 74.2 37.3 AD-521469.1 75.6 16.4 AD-521674.1 78.4 26.1 AD-521983.1 80.9 26.8 AD-521385.1 82.8 18.7 AD-521937.1 84.8 29.7 AD-522141.1 87.4 22.7 AD-522143.1 90.7 25.1 AD-521669.1 103.8 12.8

TABLE 16 PNPLA3 Single Dose Screen in Cos-7 Cells (Human Dual-Luciferase psiCHECK2 vector) 10 nM 1 nM 0.1 nM % of % of % of Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV AD-520062.3 32.2  7.2  74.3 13.0  85.9 14.6 AD-520060.1 35.5 17.3  65.8  8.7  81.0 14.5 AD-520064.3 20.4  3.3  49.2  7.7  73.6  8.0 AD-518983.1 64.3  5.2  91.2 20.1  89.9 26.0 AD-520061.1 23.0  5.7  46.1  4.9  84.5 13.2 AD-520063.2 37.5  9.8  76.0  9.4  84.4  9.9 AD-519615.1 44.2 13.4  81.4 19.8  93.9 41.4 AD-519757.1 42.0  6.3  84.4 11.8  90.2 13.3 AD-519329.1 53.6 11.7  86.2 13.3  90.2 10.7 AD-519324.1 34.9  9.9  76.4 12.0  94.6 21.5 AD-518811.1 60.5 17.1  90.0 27.0  94.6  7.0 AD-520059.1 40.9  5.0  78.3 16.3  96.9  7.7 AD-519616.1 53.2 14.0  86.4 15.9  72.7  8.3 AD-518655.1 41.3  0.5  92.0 12.2 106.1 16.9 AD-519617.1 51.8 14.3  87.1 17.2  92.2 11.4 AD-519307.1 32.3  8.7  66.2 11.2  94.9 14.7 AD-520065.3 34.1  4.1  61.3 15.7  94.1 12.5 AD-519323.1 32.0 10.9  48.0  8.9  68.7 10.9 AD-519331.1 47.8 14.6 106.6 23.3  92.0  8.0 AD-518922.1 54.3 11.2  95.1 20.4 106.8  7.7 AD-519339.1 37.6  9.6  60.4 22.4  81.9 17.8 AD-67552.2 67.3  7.3  92.6 24.3 100.9 18.4 AD-519756.1 49.8 12.6  91.6 17.1 101.2  6.3 AD-519333.1 45.1  8.0  88.4  8.4  78.8 20.1 AD-519612.1 73.4 12.1  88.1 18.7  89.4 21.7 AD-519762.1 42.0  7.3  84.9 21.9  99.1 10.2 AD-75265.3 47.2 11.9  89.2 23.8  88.3  7.9 AD-67554.5 47.8  7.3  66.0 11.0  67.2 10.6 AD-518928.1 39.2 11.0  87.7 13.2  93.2 21.4 AD-519578.1 94.4 24.3 105.6 14.6  92.6 12.0 AD-75277.3 44.7  8.3  78.5 13.7 100.4 15.2 AD-519317.1 69.1 19.2  88.7 15.7 102.9 20.2 AD-67581.5 72.9 15.9 109.6 26.6 105.3 11.4 AD-519753.1 53.1 13.6  89.6 15.3 103.0 16.2 AD-518701.1 42.9 12.5  79.1 15.0  99.8 19.4 AD-519752.1 69.5 24.6  80.7 17.2  94.1  8.3 AD-519328.1 40.9  8.5  73.8 15.7  84.9  7.7 AD-519322.1 55.7 13.6  98.3  5.1 103.9 13.1 AD-519911.1 35.0  7.9  69.8 18.3  87.2  8.7 AD-519029.1 79.1 20.1 122.2 10.1  95.6 17.3 AD-519913.1 99.4 16.9  95.1 15.7 105.5 32.2 AD-518924.1 83.2 11.9  89.6 14.5  87.4 11.7 AD-519766.1 57.7 22.1  73.9 17.8  95.2 14.4 AD-519069.1 57.9 17.5  96.2  5.7  91.2 19.5 AD-519614.1 64.9  8.9  99.4 20.3 100.9 17.1 AD-519618.1 67.5 16.8  99.9 16.1  93.5 13.3 AD-519326.1 75.6 18.3  95.8 12.7  94.8 23.2 AD-518920.1 82.8 16.5  93.8 12.4 100.8 13.4 AD-519760.1 27.5  7.6  79.0 14.0  80.3 17.9 AD-518813.1 51.7 19.6 112.4  8.9  97.9 19.1 AD-519396.1 55.9  9.5  81.0 17.7 106.8 15.1 AD-519935.1 55.7 12.5 103.7  6.8  95.0 22.2 AD-519758.1 45.5 20.8  85.1 18.2  96.4 27.4 AD-518831.1 48.2  7.4  74.1 13.5  94.7  5.8 AD-518923.1 42.9  6.0  68.5  9.3  93.0 18.0 AD-519755.2 39.5  8.8  61.5 15.2 106.9 15.0 AD-520116.1 36.8  3.8  75.9 18.4 101.4 23.0 AD-519093.1 61.5 10.0  84.2 27.7  81.5 17.9 AD-67588.2 61.3  5.7  97.6 13.0  86.5 12.2 AD-519754.3 52.3  9.4  83.0 19.3 111.7  6.7 AD-519308.1 68.0  5.3  95.7 18.5 115.4 18.7 AD-519759.1 46.6  9.8  87.3 12.5  79.0 11.5 AD-519763.1 46.3 13.4  83.5 25.2 101.6 10.6 AD-519327.1 68.6 16.9  98.2 23.1 103.5  9.0 AD-519761.1 18.5  1.5  67.3 17.5  83.3 16.3 AD-518702.1 48.1  6.4  81.8 15.2  76.4 21.1 AD-519094.1 52.5 11.0  97.8  5.3  95.7 22.7 AD-518832.1 57.1 17.0  84.6  8.0  87.6 16.3 AD-519767.1 66.8 16.3  93.7 22.7  86.8 12.2 AD-518917.1 69.6  9.7  93.3 17.5  82.3  8.7 AD-518988.1 56.8 19.5  80.0 26.5  86.4  1.6 AD-519305.1 79.5 11.9 105.5 20.8  95.4 20.1 AD-519613.1 80.4  7.8 118.8 25.0 109.9 31.4 AD-519121.1 73.3 11.7 105.3 15.4  92.5 20.7 AD-519095.1 67.5  7.3  96.6 12.7  79.1 15.9 AD-518982.1 84.1 11.8 102.3 17.9  85.5 14.6 AD-519096.1 67.1 25.2  95.0 13.2  93.0 17.4 AD-518986.1 66.1 20.6  89.7 15.6  87.7 12.2 AD-518921.1 70.9 10.9 106.0 15.7  93.2 13.3 AD-518985.1 50.1  6.9 103.5 21.2  93.9  6.5 AD-518984.1 63.4  9.0 100.8 12.9  88.6 14.2 AD-519325.1 55.5 21.1  91.3 22.8  85.2  8.4 AD-518812.1 73.5 10.8  97.7 16.5  92.5 10.4 AD-518987.1 71.8  6.9 103.7 30.5  83.8 18.6 AD-518958.1 90.6 14.1 101.6 19.1  89.7 17.2 AD-67572.2 82.4  5.9  90.5 30.4  95.9 10.6 AD-518980.1 92.4 19.0  91.3  6.0  91.2  9.6 AD-519070.1 90.6 12.2 101.4  7.7 107.6  3.4 AD-518925.1 77.4 19.4  89.6 16.7 100.6 27.1 AD-518981.1 92.4 27.7 108.2 18.0 100.8 33.9

TABLE 17 PNPLA3 Single Dose Screen in Hep3B Cells 10 nM 1 nM 0.1 nM % of % of % of Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV AD-520062.3 11.5 7.8 18.8 6.0  63.8  7.2 AD-520060.1 28.7 5.6 67.5 17.5 111.0 20.0 AD-520064.3 30.1 2.8 56.9 4.9  94.7 10.2 AD-518983.1 31.3 1.8 84.7 24.7 141.6 11.1 AD-520061.1 32.3 6.2 56.8 5.2 101.0 25.0 AD-520063.2 36.5 4.6 61.7 5.2  96.5 11.3 AD-519615.1 38.6 4.7 36.2 9.9  91.7 15.5 AD-519757.1 38.8 11.4 54.6 9.7 102.5 18.5 AD-519329.1 39.7 11.3 71.7 3.6  87.5 13.4 AD-519324.1 42.2 6.4 72.6 10.5  83.0 10.2 AD-518811.1 43.4 7.6 66.6 17.1 108.4 17.2 AD-520059.1 43.6 7.1 62.7 7.0 106.7 19.5 AD-519616.1 44.9 6.7 71.7 9.8  91.4 12.4 AD-518655.1 45.0 6.6 59.5 15.3  99.2  6.1 AD-519617.1 48.2 2.5 60.5 11.5  74.6  5.8 AD-519307.1 51 7.438 42.9 9.151  61.2  4.0 AD-520065.3 51.0 8.8 63.3 7.1 101.9 22.0 AD-519323.1 51.3 6.0 63.3 8.5 118.9 13.4 AD-519331.1 51.3 4.8 82.6 19.5  97.3 20.7 AD-518922.1 52.0 8.7 52.9 16.8  89.6 28.3 AD-519339.1 52.1 5.5 61.7 9.2  99.9 21.0 AD-67552.2 53.3 11.6 90.5 17.4  96.3 18.7 AD-519756.1 53.5 8.4 55.4 1.9  91.2  7.1 AD-519333.1 53.8 14.5 56.0 6.7  86.1 18.9 AD-519612.1 54.1 9.3 77.7 6.6 119.4 10.3 AD-519762.1 55.1 17.0 58.5 7.2  81.7  9.4 AD-75265.3 55.2 6.6 74.8 9.0 118.0 11.8 AD-67554.5 55.7 5.4 73.8 15.8  82.6  9.0 AD-518928.1 56.0 1.7 55.7 9.6  82.7 15.6 AD-519578.1 56.7 8.7 83.8 13.0  83.0 15.2 AD-75277.3 56.8 6.7 65.3 12.8  78.6 13.0 AD-519317.1 57.3 3.3 66.5 4.9  77.3  5.7 AD-67581.5 57.8 6.6 74.1 7.4  84.7  5.9 AD-519753.1 59.7 14.9 71.5 9.4  70.5 14.5 AD-518701.1 59.9 8.4 60.6 8.0  78.9  9.4 AD-519752.1 61.8 5.8 69.1 7.6  88.4 10.2 AD-519328.1 61.9 10.8 54.9 5.2 101.1 18.4 AD-519322.1 61.9 6.3 87.3 13.4  89.7  8.9 AD-519911.1 62.0 7.5 60.5 5.4  85.0 12.5 AD-519029.1 62.7 9.1 73.1 13.7  99.6 23.7 AD-519913.1 62.8 8.6 82.9 6.8 115.9 12.9 AD-518924.1 63.1 11.6 82.5 10.4  98.7 19.6 AD-519766.1 63.9 10.0 91.6 24.3 117.8 22.0 AD-519069.1 64.0 4.0 83.2 15.3 104.9 13.2 AD-519614.1 64.3 5.4 36.1 5.3 102.2 12.9 AD-519618.1 64.5 8.4 66.4 4.1  98.7 15.3 AD-519326.1 64.9 24.3 91.7 19.4 113.9 29.3 AD-518920.1 65.2 12.2 72.0 2.5 108.1 18.7 AD-519760.1 66.7 10.6 55.6 14.0 101.3 28.9 AD-518813.1 66.7 3.9 69.7 13.9  95.0 13.2 AD-519396.1 67.6 11.9 72.2 15.4  96.3 20.3 AD-519935.1 67.8 11.0 54.2 16.1  94.7 19.6 AD-519758.1 67.8 8.7 82.6 6.1 100.4 12.5 AD-518831.1 68.3 7.5 61.1 7.6 117.6 18.9 AD-518923.1 68.5 18.3 49.8 8.3  65.8  5.4 AD-519755.2 69.6 9.3 66.7 13.7  80.9 18.8 AD-520116.1 69.8 8.6 72.4 10.5  93.5 10.7 AD-519093.1 70.0 3.2 72.8 15.5 101.0 20.0 AD-67588.2 70.3 16.6 77.1 16.7  81.0 15.7 AD-519754.3 70.3 3.1 81.2 10.4  84.1  5.0 AD-519308.1 70.6 12.8 70.4 9.9  89.1  8.7 AD-519759.1 72.7 12.9 81.1 4.6  90.0  9.8 AD-519763.1 73.4 15.7 90.9 13.9 109.1 30.5 AD-519327.1 73.5 8.9 97.8 14.4 117.2 17.9 AD-519761.1 73.7 7.4 64.9 7.0 119.2 16.2 AD-518702.1 75.3 17.3 59.4 6.9  81.0  3.4 AD-519094.1 76.3 8.0 88.9 15.9  91.4  4.9 AD-518832.1 80.2 5.7 97.1 11.2 104.6 20.7 AD-519767.1 82.0 10.1 92.9 14.7 106.7 17.6 AD-518917.1 82.5 8.7 83.9 11.7 115.4 35.5 AD-518988.1 82.7 7.5 98.1 25.0  99.7 24.8 AD-519305.1 83.4 11.1 85.6 9.8  85.2 17.2 AD-519613.1 84.6 10.0 101.9  14.8  91.5  6.8 AD-519121.1 84.7 17.85 76.2 12.1  93.5 11.1 AD-519095.1 86.7 51.2 93.7 13.9 102.9  8.5 AD-518982.1 88.3 8.4 111.5  20.0 136.3 25.7 AD-519096.1 89.1 20.1 94.4 8.7  89.4 13.4 AD-518986.1 89.8 7.6 105.3  10.8 108.6 20.8 AD-518921.1 94.8 6.1 102.5  17.6 117.3 20.5 AD-518985.1 95.3 18.0 91.6 19.9 133.4 23.9 AD-518984.1 95.7 10.9 108.4  17.5  98.6 31.2 AD-519325.1 95.7 13.3 103.4  20.2 138.5 12.3 AD-518812.1 96.3 9.5 90.9 19.8 129.1 18.0 AD-518987.1 99.8 15.1 94.2 19.4  87.6 22.5 AD-518958.1 102.9 15.9 107.4  17.9  93.7  7.0 AD-67572.2 109.7 17.8 112.8  14.9 133.8 13.3 AD-518980.1 111.5 3.6 114.5  17.5 106.7 18.4 AD-519070.1 114.9 19.2 104.6  12.7 112.8 11.8 AD-518925.1 117.1 9.9 118.4  16.0  80.3  5.4 AD-518981.1 127.7 11.0 109.6  23.6  96.1 11.5

TABLE 18 PNPLA3 Single Dose Screen in Cos-7 Cells (Human Dual-Luciferase psiCHECK2 vector) 50 nM 10 nM 1 nM 0.1 nM % of % of % of % of Message ST Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEV AD-521086.1 19.5  7.9 AD-805632.1  78.2  9.4  86.1 14.1  82.9 13.9 AD-805644.1  94.2 12.3  98.0 15.8  93.5 24.2 AD-521465.1 19.2 10.1 AD-805631.1  32.8  8.2  69.8 16.2 103.4  3.9 AD-805643.1  67.1 13.2  95.1 18.0  91.9 15.7 AD-520902.1 24.1  9.4 AD-805634.1  61.6  5.2  91.7 13.2 101.0 13.3 AD-805646.1  76.3 15.6  77.5 18.6  78.0 12.5 AD-522140.1 24.3  4.9 AD-805635.1  31.1 11.5  56.5  7.6  74.8 12.2 AD-805647.1  77.9  6.4  88.6 15.9  98.2 15.2 AD-520903.1 17.6 14.3 AD-805628.1  90.5 13.5  89.6 22.6  95.7  8.1 AD-805640.1  98.8 19.0 102.0  8.7  92.8  8.1 AD-520973.1 13.0  9.6 AD-805625.1  98.8 16.3  99.8 13.9  90.4  6.5 AD-805637.1 109.0 27.2  90.5  7.5 104.5 21.2 AD-521129.1 18.9  5.7 AD-805630.1  67.5 18.8 121.7 33.2 104.0 19.0 AD-805642.1  66.1  8.1  94.9 21.9  92.2 19.3 AD-521124.1 15.6 12.7 AD-805626.1  50.3 18.2  88.4 10.9  96.6 15.9 AD-805638.1  84.8 16.6  86.4  7.5  93.1  5.4 AD-521420.1 12.6  5.2 AD-805624.1  23.0  8.7  32.9  3.5  86.6 17.4 AD-805636.1  58.4 11.6  91.3 18.9  95.1 17.6 AD-521840.1 17.8  3.3 AD-805629.1  48.7 16.6  78.6 15.6 121.6 21.4 AD-805641.1  89.0 12.5  97.2 22.4  96.1 14.5 AD-521409.1 20.5 12.1 AD-805633.1  83.0 15.2  98.6 20.5  91.8 13.6 AD-805645.1  53.2  7.4  80.7  6.3 104.6 15.8 AD-521486.1 17.6  4.6 AD-805627.1  61.1  7.9 100.3 17.8  83.7 17.9 AD-805639.1  89.7  8.6 101.3 18.2  85.1 10.0

TABLE 19 PNPLA3 Single Dose Screen in Hep3B Cells 10 nM 1 nM 0.1 nM % of % of % of Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV AD-521086.1 AD-805632.1  58.2  6.1  65.1 10.6  91.6 17.9 AD-805644.1  90.5 19.6 101.7  9.9  91.9 14.7 AD-521465.1 AD-805631.1  54.6  4.6  81.2  9.9 102.5 13.4 AD-805643.1  64.0 11.6  80.5 23.2  92.2  8.3 AD-520902.1 AD-805634.1 102.2 18.8  88.4  4.6 113.2 20.7 AD-805646.1  95.8 12.0  95.3  5.6  81.5  8.5 AD-522140.1 AD-805635.1  90.2 10.5  52.8  6.7  84.9 14.4 AD-805647.1  96.5 24.1  73.6  4.3  83.4 11.4 AD-520903.1 AD-805628.1 105.2 25.9 106.7  6.8 101.7 16.1 AD-805640.1 103.1  6.2  97.8 13.0  91.9  9.5 AD-520973.1 AD-805625.1 109.4 11.7 112.1 21.2 109.8 21.6 AD-805637.1 123.9 17.4 112.6 13.6 108.7 15.7 AD-521129.1 AD-805630.1 105.9  6.0 109.6 13.9 121.5 16.2 AD-805642.1 109.0 19.2  91.3 11.0 110.5 13.3 AD-521124.1 AD-805626.1  74.6  8.2  87.8 10.7 112.2 10.1 AD-805638.1  98.0  9.2  97.9  6.4 104.3 13.6 AD-521420.1 AD-805624.1  33.7  5.4  42.4  9.2  53.2  6.9 AD-805636.1  82.1  7.5  89.7  8.8  95.7 11.6 AD-521840.1 AD-805629.1  60.7  5.7  82.3 17.7  93.5  5.7 AD-805641.1 122.2 21.4 115.9 21.0 111.4  9.3 AD-521409.1 AD-805633.1 116.6 19.7 107.0 24.0 127.8 17.3 AD-805645.1  96.6  9.7  89.0 11.0  90.9 13.2 AD-521486.1 AD-805627.1  56.3  6.9  78.2 14.1 115.9 10.4 AD-805639.1 117.8 13.8 112.2  9.7  94.7  9.5

Example 3. In Vivo Screening of dsRNA Duplexes in Mice

Duplexes of interest, identified from the above in vitro studies, were evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹¹ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding the open reading frame and 3′ UTR of human PNPLA3 mRNA referenced as NM_025225.2 (AAV8.-TBG-PI-PNPLA3).

At day 0, groups of three mice were subcutaneously administered a single 3 mg/kg dose or a single 10 mg/kg of the agents of interest or PBS control. Table 20 provides the treatment groups and Table 21 provides the modified nucleotide sequences of the sense and antisense strands of the duplexes of interest. At day 7 or day 14 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Tissue mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to housekeeping gene GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 22 and shown in FIG. 1, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 20 Group Animal Timepoint # # Treatment Dose (Day) 1 1 PBS n/a D7  2 3 2 4 Naïve n/a 5 6 3 7 AD-517197.2 3 mpk 8 9 4 10 AD-517258.2 11 12 5 13 AD-517197.2 10 mpk 14 15 6 16 AD-517258.2 17 18 7 19 PBS n/a D14 20 21 8 22 Naïve n/a 23 24 9 25 AD-517197.2 3 mpk 26 27 10 28 AD-517258.2 29 30 11 31 AD-517197.2 10 mpk 32 33 12 34 AD-517258.2 35 36

TABLE 21 SEQ ID Duplex ID Oligo ID Strand Nucleotide Sequence 5′ to 3′ NO: AD-517197.2 A-150304.3 sense csasugagCfaAfGfAfuuugcaacuuL96 1746 A-999710.1 antis asAfsguug(Cgn)aaaucuUfgCfucaugsusa 1747 AD-517258.2 A-999829.1 sense ascsuugcUfaCfCfCfauuaggauauL96 1748 A-999830.1 antis asUfsaucc(Tgn)aaugggUfaGfcaagususg 1749

TABLE 22 % Message Remaining Duplex Normalized to PBS SD PBS Day 7 101.19 11.20 Naïve Day 7 89.04 18.18 AD-517197.2 3 mpk 84.36 5.65 AD-517258 3 mpk 76.75 12.42 AD-517197.2 10 mpk 56.72 6.90 AD-517258 10 mpk 77.23 10.55 PBS Day 14 103.60 19.54 Naïve Day 14 95.55 14.11 AD-517197.2 3 mpk 89.05 3.84 AD-517258 3 mpk 136.33 11.85 AD-517197.2 10 mpk 78.79 9.48 AD-517258 10 mpk 61.83 6.29

Additional duplexes of interest, identified from the above in vitro studies, were also evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹¹ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding the open reading frame and the 3′ UTR of human PNPLA3 referenced in NM_025225.2 (AAV8.-TBG-PI-PNPLA3).

At day 0, time 0, groups of three mice were subcutaneously administered a single 3 mg/kg dose of an agent of interest (with the exception of AD-517258.2) which was followed about 1 hour later by a single 7 mg/kg dose of the same agent administered at time 0, or PBS control at day 0, time 0. Mice administered AD-517258.2 were only administered a single 3 mg/kg dose at day 0, time 0. Table 20 provides the treatment groups and Table 21 provides the modified nucleotide sequences of the sense and antisense strands of the duplexes of interest. At day 7 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Tissue mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to housekeeping gene GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 25 and shown in FIG. 2, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 23 Group # Animal # Treatment Dose 1 1 PBS n/a 2 3 2 4 Naïve n/a 5 6 3 7 AD-517197.2 10 mpk (3 mpk followed 8 by 7 mpk ~1 hr later) 9 4 10 AD-516748.2 11 12 5 13 AD-516851.2 14 15 6 16 AD-517258.2 3 mpk 17 18 7 19 AD-519351.2 10 mpk (3 mpk followed 20 by 7 mpk ~1 hr later) 21 8 22 AD-519754.2 23 24 9 25 AD-519828.2 26 27 10 28 AD-520018.2 29 30 11 31 AD-520035.2 32 33 12 34 AD-520062.2 35 36 13 37 AD-520064.2 38 39 14 40 AD-520065.2 41 42 15 43 AD-520067.2 44 45 16 46 AD-75289.2 47 48 17 49 AD-520069.2 50 51 18 52 AD-520099.2 53 54 19 55 AD-67575.7 56 57 20 58 AD-520101.2 59 60 21 61 AD-67605.7 62 63

TABLE 24 SEQ ID Duplex ID Oligo ID Strand NO: Nucleotide Sequence 5′ to 3′ AD-517197.2 A-150304.3 sense 1750 csasugagCfaAfGfAfuuugcaacuuL96 A-999710.1 antis 1751 asAfsguug(Cgn)aaaucuUfgCfucaugsusa AD-516748.2 A-998818.1 sense 1752 asasagacGfaAfGfUfcguggaugcuL96 A-998819.1 antis 1753 asGfscauc(Cgn)acgacuUfcGfucuuusgsg AD-516851.2 A-999023.1 sense 1754 usgsaugcCfaAfAfAfcaaccaucauL96 A-999024.1 antis 1755 asUfsgaug(Ggn)uuguuuUfgGfcaucasasu AD-517258.2 A-999829.1 sense 1756 ascsuugcUfaCfCfCfauuaggauauL96 A-999830.1 antis 1757 asUfsaucc(Tgn)aaugggUfaGfcaagususg AD-519351.2 A-999855.1 sense 1758 asgsgauaAfuGfUfCfuuauguaauuL96 A-1003070.1 antis 1759 asAfsuuaCfaUfAfagacAfuUfauccusasa AD-519754.2 A-1000664.1 sense 1760 gsasgcugAfgUfUfGfguuuuaugauL96 A-1003473.1 antis 1761 asUfscauAfaAfAfccaaCfuCfagcucsasg AD-519828.2 A-1000812.1 sense 1762 csasucagCfaUfGfCfguuaauucauL96 A-1003547.1 antis 1763 asUfsgaaUfuAfAfcgcaUfgCfugaugsusa AD-520018.2 A-1001193.1 sense 1764 gsasuaacCfuUfGfAfcuacuaaaauL96 A-1003737.1 antis 1765 asUfsuuuAfgUfAfgucaAfgGfuuaucsasu AD-520035.2 A-1001228.1 sense 1766 gsgsuaacAfaGfAfUfgauaaucuauL96 A-1003754.1 antis 1767 asUfsagaUfuAfUfcaucUfuGfuuaccscsc AD-520062.2 A-1001282.1 sense 1768 ascscuuuUfuCfAfCfcuaacuaaauL96 A-1003781.1 antis 1769 asUfsuuaGfuUfAfggugAfaAfaaggusgsu AD-520064.2 A-1001286.1 sense 1770 csusuuuuCfaCfCfUfaacuaaaauuL96 A-1003783.1 antis 1771 asAfsuuuUfaGfUfuaggUfgAfaaaagsgsu AD-520065.2 A-1001288.1 sense 1772 ususuuucAfcCfUfAfacuaaaauauL96 A-1003784.1 antis 1773 asUfsauuUfuAfGfuuagGfuGfaaaaasgsg AD-520067.2 A-1001292.1 sense 1774 ususucacCfuAfAfCfuaaaauaauuL96 A-1003786.1 antis 1775 asAfsuuaUfuUfUfaguuAfgGfugaaasasa AD-75289.2 A-150350.2 sense 1776 ususcaccUfaAfCfUfaaaauaauguL96 A-150351.2 antis 1777 asCfsauuAfuUfUfuaguUfaGfgugaasasa AD-520069.2 A-1001296.1 sense 1778 csasccuaAfcUfAfAfaauaauguuuL96 A-1003788.1 antis 1779 asAfsacaUfuAfUfuuuaGfuUfaggugsasa AD-520099.2 A-1001362.1 sense 1780 usgsuuacCfuGfUfUfgaauuuuguuL96 A-1003818.1 antis 1781 asAfscaaAfaUfUfcaacAfgGfuaacasasc AD-67575.7 A-135333.4 sense 1782 ususaccuGfuUfGfAfauuuuguauuL96 A-135334.4 antis 1783 asAfsuacAfaAfAfuucaAfcAfgguaascsa AD-520101.2 A-1001366.1 sense 1784 usasccugUfuGfAfAfuuuuguauuuL96 A-1003820.1 antis 1785 asAfsauaCfaAfAfauucAfaCfagguasasc AD-67605.7 A-135397.4 sense 1786 ascscuguUfgAfAfUfuuuguauuauL96 A-135398.4 antis 1787 asUfsaauAfcAfAfaauuCfaAfcaggusasa

TABLE 25 Duplex % message remaining SD PBS 103.93 20.88 Naïve 98.96 8.18 AD-517197.2 72.73 5.25 AD-516748.2 113.80 50.63 AD-516851.2 90.77 10.59 AD-517258.2 106.22 12.98 AD-519351.2 51.51 6.40 AD-519754.2 52.67 3.53 AD-519828.2 80.26 9.81 AD-520018.2 35.16 14.27 AD-520035.2 84.43 13.85 AD-520062.2 55.69 6.76 AD-520064.2 101.98 17.12 AD-520065.2 83.81 26.80 AD-520067.2 77.07 13.29 AD-75289.2 85.71 16.74 AD-520069.2 88.70 10.81 AD-520099.2 104.33 28.27 AD-67575.7 97.80 18.17 AD-520101.2 97.77 6.29 AD-67605.7 107.86 21.32

Additional duplexes of interest, identified from the above in vitro studies, were also evaluated in vivo in an AAV titration study. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹¹, 2×10¹⁰, 2×10⁹, or 2×10⁸ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding a portion of human PNPLA3 mRNA encoding the open reading frame and 3′ UTR of human PNPLA3 mRNA referenced as NM_025225.2 (AAV8.-TBG-PI-PNPLA3).

At day 0, time 0, groups of three mice were subcutaneously administered a single 10 mg/kg dose of an agent of interest or PBS control. Table 26 provides the treatment groups and Table 27 provides the modified nucleotide sequences of the sense and antisense strands of the duplexes of interest. At day 7 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Tissue mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to housekeeping gene GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 28 and shown in FIG. 3, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 26 AAV Titer Group # Animal # Treatment Dose (vp/mouse) 1 1 PBS n/a 2.00E+11 2 3 2 4 AD-519351.4 10 mpk 5 6 3 7 AD-519754.5 8 9 4 10 AD-520062.5 11 12 5 13 AD-520018.6 14 15 6 16 PBS n/a 2.00E+10 17 18 7 19 AD-519351.4 10 mpk 20 21 8 22 AD-519754.5 23 24 9 25 AD-520062.5 26 27 10 28 AD-520018.6 29 30 11 31 PBS n/a 2.00E+09 32 33 12 34 AD-519351.4 10 mpk 35 36 13 37 AD-519754.5 38 39 14 40 AD-520062.5 41 42 15 43 AD-520018.6 44 45 16 46 PBS n/a 2.00E+08 47 48 17 49 AD-519351.4 10 mpk 50 51 18 52 AD-519754.5 53 54 19 55 AD-520062.5 56 57 20 58 AD-520018.6 59 60

TABLE 27 SEQ Duplex ID Oligo ID Strand Nucleotide Sequence 5′ to 3′ ID NO: AD-519351.4 A-999855.2 sense asgsgauaAfuGfUfCfuuauguaauuL96 1788 A-1003070.2 antis asAfsuuaCfaUfAfagacAfuUfauccusasa 1789 AD-519754.5 A-1000664.3 sense gsasgcugAfgUfUfGfguuuuaugauL96 1790 A-1003473.3 antis asUfscauAfaAfAfccaaCfuCfagcucsasg 1791 AD-520062.5 A-1001282.3 sense ascscuuuUfuCfAfCfcuaacuaaauL96 1792 A-1003781.3 antis asUfsuuaGfuUfAfggugAfaAfaaggusgsu 1793 AD-520018.6 A-1001193.3 sense gsasuaacCfuUfGfAfcuacuaaaauL96 1794 A-1003737.3 antis asUfsuuuAfgUfAfgucaAfgGfuuaucsasu 1795

TABLE 28 AAV dose titer Experimental treatment Average Fold Change SD 2E11 PBS 1.02 0.25 AD-520018 0.34 0.01 AD-519351 0.63 0.09 AD-519754 0.58 0.09 AD-520062 0.51 0.17 2E10 PBS 1.09 0.49 AD-520018 0.37 0.07 AD-519351 0.64 0.15 AD-519754 0.39 0.17 AD-520062 0.37 0.18 2E9  PBS 1.02 0.23 AD-520018 0.19 0.13 AD-519351 0.30 0.16 AD-519754 0.38 0.02 AD-520062 0.18 0.16 2E8  PBS 1.05 0.45 AD-520018 0.59 0.50 AD-519351 0.74 0.35 AD-519754 1.23 0.52 AD-520062 0.87 0.10

Additional duplexes of interest, identified from the above in vitro studies, were also evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹¹ viral particles (corresponding to 2.4×10¹³ genome copies/mi) of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding a portion of human PNPLA3 mLRNA encoding the open reading frame and 3′ UTR of human PNPLA3 mLRNA referenced as NM_025225.2 (AAV8.-TBG-PI-PNPLA3).

At day 0, one group of three mice for each agent of interest was subcutaneously administered a single 10 mg/kg dose of the agent of interest or PBS control. Another group of three mice for each agent of interest was subcutaneously administered a single 10 mg/kg dose of the agent of interest or PBS control at Day 0 and a subsequent 10 mg/kg dose of the agent at Day 1. A third group of three mice for each agent of interest was subcutaneously administered a single 10 mg/kg dose of the agent of interest or PBS control at Day 0, a subsequent 10 mg/kg dose of the agent at Day 1, and a further 10 mg/kg dose of the agent at Day 2. Table 29 provides the treatment groups and Table 30 provides the modified nucleotide sequences of the sense and antisense strands of the duplexes of interest. Seven days after the last dose was administered, animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Tissue mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to housekeeping gene GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 31 and shown in FIG. 4, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 29 Group Animal End of # # Treatment Dose Study 1 1 PBS n/a D7 2 3 2 4 naïve n/a 5 6 3 7 AD-520018.2 10 mpk D0 8 9 4 10 AD-519351.2 11 12 5 13 AD-519754.2 14 15 6 16 AD-520062.2 17 18 7 19 PBS 10 mpk D0, D8 20 10 mpk D1 21 (20 mpk total) 8 22 AD-520018.2 23 24 9 25 AD-519351.2 26 27 10 28 AD-519754.2 29 30 11 31 AD-520062.2 32 33 12 34 PBS 10 mpk D0, D9 35 10 mpk D1, 36 10 mpk D2 13 37 AD-520018.2 (30 mpk total) 38 39 14 40 AD-519351.2 41 42 15 43 AD-519754.2 44 45 16 46 AD-520062.2 47 48

TABLE 30 Duplex ID Oligo ID Strand Nucleotide Sequence 5′ to 3′ SEQ ID NO: AD-520018.2 A-1001193.1 sense gsasuaacCfuUfGfAfcuacuaaaauL96 1796 A-1003737.1 antis asUfsuuuAfgUfAfgucaAfgGfuuaucsasu 1797 AD-519351.2 A-999855.1 sense asgsgauaAfuGfUfCfuuauguaauuL96 1798 A-1003070.1 antis asAfsuuaCfaUfAfagacAfuUfauccusasa 1799 AD-519754.2 A-1000664.1 sense gsasgcugAfgUfUfGfguuuuaugauL96 1800 A-1003473.1 antis asUfscauAfaAfAfccaaCfuCfagcucsasg 1801 AD-520062.2 A-1001282.1 sense ascscuuuUfuCfAfCfcuaacuaaauL96 1802 A-1003781.1 antis asUfsuuaGfuUfAfggugAfaAfaaggusgsu 1803

TABLE 31 % message Duplex remaining SD PBS 101.3 18.9 10 mpk D7 Naive 120.9 24.9 10 mpk D7 AD-520018.2 10 mpk D7 41.7 12.3 AD-519351.2 10 mpk D7 35.9 9.4 AD-519754.2 10 mpk D7 58.6 6.8 AD-520062.2 10 mpk D7 45.4 6.4 PBS 100.6 12.4 20 mpk D8 AD-520018.2 20mpk D8 31.8 5.6 AD-519351.2 20mpk D8 27.9 1 AD-519754.2 20mpk D 50.5 4.3 AD-520062.2 20mpk D8 49.9 4.3 PBS 101.8 23.3 30 mpk D9 AD-520018.2 30mpk D9 45.8 18.5 AD-519351.2 30mpk D9 22.4 10.4 AD-519754.2 30mpk D9 41.3 9.5 AD-520062.2 30mpk D9 61.4 12.3

Example 4. Design, Synthesis and In Vitro Screening of Additional dsRNA Duplexes

Additional siRNAs were designed, synthesized, and prepared using methods known in the art and described above in Example 1.

Detailed lists of the additional unmodified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 32. Detailed lists of the modified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 33.

For transfections, cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 7.5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 2.5 μl of each siRNA duplex to an individual well in a 384-well plate. The mixture was then incubated at room temperature for 15 minutes. Forty μl of complete growth media without antibiotic containing ˜1.5×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 50, nM, 10 nM, 1 nM, and 0.1 nM final duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 3 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10× dNTPs, 1.51 Random primers, 0.751 Reverse Transcriptase, 0.751 RNase inhibitor and 9.91 of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

RT-qPCR was performed as described above and relative fold change was calculated as described above.

The results of the transfection assays of the dsRNA agents listed in Tables 32 and 33 in Hep3B cells are shown in Table 34. The results of the transfection assays of the dsRNA agents listed in Tables 32 and 33 in primary cynomolgus hepatocytes (PCH) are shown in Table 35.

TABLE 32 Unmodified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ SEQ ID Range in Antisense ID Range in Duplex Name Sense Sequence 5′to 3′ NO: NM_025225.2 Sequence 5′to 3′ NO: NM_025225.2 AD-518658.1 GUCCUCUCAGAUCUUGUGCGU 1804 375-395 ACGCACAAGAUCUGAGAGGACCU 1938 373-395 AD-518707.1 CAUCCAUCCUUCAACUUAAGU 1805 426-446 ACUUAAGUUGAAGGAUGGAUGGA 1939 424-446 AD-518708.1 AUCCAUCCUUCAACUUAAGCU 1806 427-447 AGCUUAAGUUGAAGGAUGGAUGG 1940 425-447 AD-518709.1 UCCAUCCUUCAACUUAAGCAU 1807 428-448 AUGCUUAAGUUGAAGGAUGGAUG 1941 426-448 AD-518722.1 UUAAGCAAGUUCCUCCGACAU 1808 441-461 AUGUCGGAGGAACUUGCUUAAGU 1942 439-461 AD-518789.1 GCAAAAUAGGCAUCUCUCUUU 1809 508-528 AAAGAGAGAUGCCUAUUUUGCCG 1943 506-528 AD-518791.1 AAAAUAGGCAUCUCUCUUACU 1810 510-530 AGUAAGAGAGAUGCCUAUUUUGC 1944 508-530 AD-518792.1 AAAUAGGCAUCUCUCUUACCU 1811 511-531 AGGUAAGAGAGAUGCCUAUUUUG 1945 509-531 AD-518794.1 AUAGGCAUCUCUCUUACCAGU 1812 513-533 ACUGGUAAGAGAGAUGCCUAUUU 1946 511-533 AD-518800.1 AUCUCUCUUACCAGAGUGUCU 1813 519-539 AGACACUCUGGUAAGAGAGAUGC 1947 517-539 AD-518827.1 ACUUUCGGUCCAAAGACGAAU 1814 565-585 AUUCGUCUUUGGACCGAAAGUCA 1948 563-585 AD-518829.1 UUUCGGUCCAAAGACGAAGUU 1815 567-587 AACUUCGUCUUUGGACCGAAAGU 1949 565-587 AD-518831.2 CGGUCCAAAGACGAAGUCGUU 1816 570-590 AACGACUUCGUCUUUGGACCGAA 1950 568-590 AD-518832.2 GGUCCAAAGACGAAGUCGUGU 1817 571-591 ACACGACUUCGUCUUUGGACCGA 1951 569-591 AD-518857.1 UUGGUAUGUUCCUGCUUCAUU 1818 597-617 AAUGAAGCAGGAACAUACCAAGG 1952 595-617 AD-518921.2 UGACAACGUACCCUUCAUUGU 1819 683-703 ACAAUGAAGGGUACGUUGUCACU 1953 681-703 AD-518922.2 GACAACGUACCCUUCAUUGAU 1820 684-704 AUCAAUGAAGGGUACGUUGUCAC 1954 682-704 AD-518923.2 ACAACGUACCCUUCAUUGAUU 1821 685-705 AAUCAAUGAAGGGUACGUUGUCA 1955 683-705 AD-518924.2 CAACGUACCCUUCAUUGAUGU 1822 686-706 ACAUCAAUGAAGGGUACGUUGUC 1956 684-706 AD-518925.2 AACGUACCCUUCAUUGAUGCU 1823 687-707 AGCAUCAAUGAAGGGUACGUUGU 1957 685-707 AD-518967.1 UAAAGUCAAGUCCACGAACUU 1824 758-778 AAGUUCGUGGACUUGACUUUAGG 1958 756-778 AD-518968.1 AAAGUCAAGUCCACGAACUUU 1825 759-779 AAAGUUCGUGGACUUGACUUUAG 1959 757-779 AD-518976.1 UCCACGAACUUUCUUCAUGUU 1826 768-788 AACAUGAAGAAAGUUCGUGGACU 1960 766-788 AD-518977.1 CCACGAACUUUCUUCAUGUGU 1827 769-789 ACACAUGAAGAAAGUUCGUGGAC 1961 767-789 AD-518978.1 CACGAACUUUCUUCAUGUGGU 1828 770-790 ACCACAUGAAGAAAGUUCGUGGA 1962 768-790 AD-518979.1 ACGAACUUUCUUCAUGUGGAU 1829 771-791 AUCCACAUGAAGAAAGUUCGUGG 1963 769-791 AD-518980.2 CGAACUUUCUUCAUGUGGACU 1830 772-792 AGUCCACAUGAAGAAAGUUCGUG 1964 770-792 AD-519043.1 UUCUCUCGAGAGCUUUUGUCU 1831 835-855 AGACAAAAGCUCUCGAGAGAAGG 1965 833-855 AD-519340.1 UGCUACCCAUUAGGAUAAUGU 1832 1207-1227 ACAUUAUCCUAAUGGGUAGCAAG 1966 1205-1227 AD-519341.1 GCUACCCAUUAGGAUAAUGUU 1833 1208-1228 AACAUUAUCCUAAUGGGUAGCAA 1967 1206-1228 AD-519342.1 CUACCCAUUAGGAUAAUGUCU 1834 1209-1229 AGACAUUAUCCUAAUGGGUAGCA 1968 1207-1229 AD-519343.1 UACCCAUUAGGAUAAUGUCUU 1835 1210-1230 AAGACAUUAUCCUAAUGGGUAGC 1969 1208-1230 AD-519344.1 ACCCAUUAGGAUAAUGUCUUU 1836 1211-1231 AAAGACAUUAUCCUAAUGGGUAG 1970 1209-1231 AD-519349.1 UUAGGAUAAUGUCUUAUGUAU 1837 1216-1236 AUACAUAAGACAUUAUCCUAAUG 1971 1214-1236 AD-519350.1 UAGGAUAAUGUCUUAUGUAAU 1838 1217-1237 AUUACAUAAGACAUUAUCCUAAU 1972 1215-1237 AD-519477.1 UCACAGGUGUUCACUCGAGUU 1839 1344-1364 AACUCGAGUGAACACCUGUGAGG 1973 1342-1364 AD-519596.1 CUUGGGCAAUAAAGUACCUGU 1840 1544-1564 ACAGGUACUUUAUUGCCCAAGAA 1974 1542-1564 AD-519622.1 UUCCCAGUUUUUCACUAGAGU 1841 1588-1608 ACUCUAGUGAAAAACUGGGAAAG 1975 1586-1608 AD-519666.1 GGCGAGUCUAGCAGAUUCUUU 1842 1632-1652 AAAGAAUCUGCUAGACUCGCCUC 1976 1630-1652 AD-519667.1 GCGAGUCUAGCAGAUUCUUUU 1843 1633-1653 AAAAGAAUCUGCUAGACUCGCCU 1977 1631-1653 AD-519668.1 CGAGUCUAGCAGAUUCUUUCU 1844 1634-1654 AGAAAGAAUCUGCUAGACUCGCC 1978 1632-1654 AD-519669.1 GAGUCUAGCAGAUUCUUUCAU 1845 1635-1655 AUGAAAGAAUCUGCUAGACUCGC 1979 1633-1655 AD-519670.1 AGUCUAGCAGAUUCUUUCAGU 1846 1636-1656 ACUGAAAGAAUCUGCUAGACUCG 1980 1634-1656 AD-519671.1 GUCUAGCAGAUUCUUUCAGAU 1847 1637-1657 AUCUGAAAGAAUCUGCUAGACUC 1981 1635-1657 AD-519672.1 UCUAGCAGAUUCUUUCAGAGU 1848 1638-1658 ACUCUGAAAGAAUCUGCUAGACU 1982 1636-1658 AD-519691.1 UGCUAAAGUUUCCCAUCUUUU 1849 1659-1679 AAAAGAUGGGAAACUUUAGCACC 1983 1657-1679 AD-519692.1 GCUAAAGUUUCCCAUCUUUGU 1850 1660-1680 ACAAAGAUGGGAAACUUUAGCAC 1984 1658-1680 AD-519693.1 CUAAAGUUUCCCAUCUUUGUU 1851 1661-1681 AACAAAGAUGGGAAACUUUAGCA 1985 1659-1681 AD-519694.1 UAAAGUUUCCCAUCUUUGUGU 1852 1662-1682 ACACAAAGAUGGGAAACUUUAGC 1986 1660-1682 AD-519696.1 AAGUUUCCCAUCUUUGUGCAU 1853 1664-1684 AUGCACAAAGAUGGGAAACUUUA 1987 1662-1684 AD-67554.6 UCUGAGCUGAGUUGGUUUUAU 1854 1740-1760 AUAAAACCAACUCAGCUCAGAGG 1988 1738-1760 AD-519752.2 CUGAGCUGAGUUGGUUUUAUU 1855 1741-1761 AAUAAAACCAACUCAGCUCAGAG 1989 1739-1761 AD-519753.2 UGAGCUGAGUUGGUUUUAUGU 1856 1742-1762 ACAUAAAACCAACUCAGCUCAGA 1990 1740-1762 AD-519754.8 GAGCUGAGUUGGUUUUAUGAU 1857 1743-1763 AUCAUAAAACCAACUCAGCUCAG 1991 1741-1763 AD-519755.4 AGCUGAGUUGGUUUUAUGAAU 1858 1744-1764 AUUCAUAAAACCAACUCAGCUCA 1992 1742-1764 AD-519759.2 GAGUUGGUUUUAUGAAAAGCU 1859 1748-1768 AGCUUUUCAUAAAACCAACUCAG 1993 1746-1768 AD-519760.2 AGUUGGUUUUAUGAAAAGCUU 1860 1749-1769 AAGCUUUUCAUAAAACCAACUCA 1994 1747-1769 AD-519761.2 GUUGGUUUUAUGAAAAGCUAU 1861 1750-1770 AUAGCUUUUCAUAAAACCAACUC 1995 1748-1770 AD-519766.2 UUUUAUGAAAAGCUAGGAAGU 1862 1755-1775 ACUUCCUAGCUUUUCAUAAAACC 1996 1753-1775 AD-519773.1 AAAAGCUAGGAAGCAACCUUU 1863 1762-1782 AAAGGUUGCUUCCUAGCUUUUCA 1997 1760-1782 AD-519776.1 AGCUAGGAAGCAACCUUUCGU 1864 1765-1785 ACGAAAGGUUGCUUCCUAGCUUU 1998 1763-1785 AD-519777.1 GCUAGGAAGCAACCUUUCGCU 1865 1766-1786 AGCGAAAGGUUGCUUCCUAGCUU 1999 1764-1786 AD-519779.1 UAGGAAGCAACCUUUCGCCUU 1866 1768-1788 AAGGCGAAAGGUUGCUUCCUAGC 2000 1766-1788 AD-519809.1 CCAGCACUUAACUCUAAUACU 1867 1798-1818 AGUAUUAGAGUUAAGUGCUGGAC 2001 1796-1818 AD-519810.1 CAGCACUUAACUCUAAUACAU 1868 1799-1819 AUGUAUUAGAGUUAAGUGCUGGA 2002 1797-1819 AD-519811.1 AGCACUUAACUCUAAUACAUU 1869 1800-1820 AAUGUAUUAGAGUUAAGUGCUGG 2003 1798-1820 AD-519814.1 ACUUAACUCUAAUACAUCAGU 1870 1803-1823 ACUGAUGUAUUAGAGUUAAGUGC 2004 1801-1823 AD-519820.1 CUCUAAUACAUCAGCAUGCGU 1871 1809-1829 ACGCAUGCUGAUGUAUUAGAGUU 2005 1807-1829 AD-519821.1 UCUAAUACAUCAGCAUGCGUU 1872 1810-1830 AACGCAUGCUGAUGUAUUAGAGU 2006 1808-1830 AD-519822.1 CUAAUACAUCAGCAUGCGUUU 1873 1811-1831 AAACGCAUGCUGAUGUAUUAGAG 2007 1809-1831 AD-519823.1 UAAUACAUCAGCAUGCGUUAU 1874 1812-1832 AUAACGCAUGCUGAUGUAUUAGA 2008 1810-1832 AD-519826.1 UACAUCAGCAUGCGUUAAUUU 1875 1815-1835 AAAUUAACGCAUGCUGAUGUAUU 2009 1813-1835 AD-519827.1 ACAUCAGCAUGCGUUAAUUCU 1876 1816-1836 AGAAUUAACGCAUGCUGAUGUAU 2010 1814-1836 AD-519828.3 CAUCAGCAUGCGUUAAUUCAU 1877 1817-1837 AUGAAUUAACGCAUGCUGAUGUA 2011 1815-1837 AD-519830.1 UCAGCAUGCGUUAAUUCAGCU 1878 1819-1839 AGCUGAAUUAACGCAUGCUGAUG 2012 1817-1839 AD-519832.1 AGCAUGCGUUAAUUCAGCUGU 1879 1821-1841 ACAGCUGAAUUAACGCAUGCUGA 2013 1819-1841 AD-519887.1 GUCCCUUACUGACUGUUUCGU 1880 1876-1896 ACGAAACAGUCAGUAAGGGACCC 2014 1874-1896 AD-519888.1 UCCCUUACUGACUGUUUCGUU 1881 1877-1897 AACGAAACAGUCAGUAAGGGACC 2015 1875-1897 AD-519889.1 CCCUUACUGACUGUUUCGUGU 1882 1878-1898 ACACGAAACAGUCAGUAAGGGAC 2016 1876-1898 AD-519890.1 CCUUACUGACUGUUUCGUGGU 1883 1879-1899 ACCACGAAACAGUCAGUAAGGGA 2017 1877-1899 AD-519929.1 UUCCAGCAUGAGGUUCUUAGU 1884 1919-1939 ACUAAGAACCUCAUGCUGGAACA 2018 1917-1939 AD-519931.1 CCAGCAUGAGGUUCUUAGAAU 1885 1921-1941 AUUCUAAGAACCUCAUGCUGGAA 2019 1919-1941 AD-519932.1 CAGCAUGAGGUUCUUAGAAUU 1886 1922-1942 AAUUCUAAGAACCUCAUGCUGGA 2020 1920-1942 AD-519933.1 AGCAUGAGGUUCUUAGAAUGU 1887 1923-1943 ACAUUCUAAGAACCUCAUGCUGG 2021 1921-1943 AD-519935.2 CAUGAGGUUCUUAGAAUGACU 1888 1925-1945 AGUCAUUCUAAGAACCUCAUGCU 2022 1923-1945 AD-519942.1 UUCUUAGAAUGACAGGUGUUU 1889 1932-1952 AAACACCUGUCAUUCUAAGAACC 2023 1930-1952 AD-520005.1 GGUCUGCAAAGAUGAUAACCU 1890 2097-2117 AGGUUAUCAUCUUUGCAGACCAC 2024 2095-2117 AD-520006.1 GUCUGCAAAGAUGAUAACCUU 1891 2098-2118 AAGGUUAUCAUCUUUGCAGACCA 2025 2096-2118 AD-520007.1 UCUGCAAAGAUGAUAACCUUU 1892 2099-2119 AAAGGUUAUCAUCUUUGCAGACC 2026 2097-2119 AD-520008.1 CUGCAAAGAUGAUAACCUUGU 1893 2100-2120 ACAAGGUUAUCAUCUUUGCAGAC 2027 2098-2120 AD-520011.1 CAAAGAUGAUAACCUUGACUU 1894 2103-2123 AAGUCAAGGUUAUCAUCUUUGCA 2028 2101-2123 AD-520020.1 UAACCUUGACUACUAAAAACU 1895 2112-2132 AGUUUUUAGUAGUCAAGGUUAUC 2029 2110-2132 AD-520021.1 AACCUUGACUACUAAAAACGU 1896 2113-2133 ACGUUUUUAGUAGUCAAGGUUAU 2030 2111-2133 AD-520022.1 ACCUUGACUACUAAAAACGUU 1897 2114-2134 AACGUUUUUAGUAGUCAAGGUUA 2031 2112-2134 AD-520023.1 CCUUGACUACUAAAAACGUCU 1898 2115-2135 AGACGUUUUUAGUAGUCAAGGUU 2032 2113-2135 AD-520024.1 CUUGACUACUAAAAACGUCUU 1899 2116-2136 AAGACGUUUUUAGUAGUCAAGGU 2033 2114-2136 AD-520025.1 UUGACUACUAAAAACGUCUCU 1900 2117-2137 AGAGACGUUUUUAGUAGUCAAGG 2034 2115-2137 AD-520034.1 GGGUAACAAGAUGAUAAUCUU 1901 2145-2165 AAGAUUAUCAUCUUGUUACCCCC 2035 2143-2165 AD-520035.3 GGUAACAAGAUGAUAAUCUAU 1902 2146-2166 AUAGAUUAUCAUCUUGUUACCCC 2036 2144-2166 AD-520036.1 GUAACAAGAUGAUAAUCUACU 1903 2147-2167 AGUAGAUUAUCAUCUUGUUACCC 2037 2145-2167 AD-520037.1 UAACAAGAUGAUAAUCUACUU 1904 2148-2168 AAGUAGAUUAUCAUCUUGUUACC 2038 2146-2168 AD-520038.1 AACAAGAUGAUAAUCUACUUU 1905 2149-2169 AAAGUAGAUUAUCAUCUUGUUAC 2039 2147-2169 AD-520053.1 UUUUAGAACACCUUUUUCACU 1906 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 2040 2169-2191 AD-520060.3 ACACCUUUUUCACCUAACUAU 1907 2178-2198 AUAGUUAGGUGAAAAAGGUGUUC 2041 2176-2198 AD-520061.3 CACCUUUUUCACCUAACUAAU 1908 2179-2199 AUUAGUUAGGUGAAAAAGGUGUU 2042 2177-2199 AD-520064.5 CUUUUUCACCUAACUAAAAUU 1909 2182-2202 AAUUUUAGUUAGGUGAAAAAGGU 2043 2180-2202 AD-520065.4 UUUUUCACCUAACUAAAAUAU 1910 2183-2203 AUAUUUUAGUUAGGUGAAAAAGG 2044 2181-2203 AD-520066.2 UUUUCACCUAACUAAAAUAAU 1911 2184-2204 AUUAUUUUAGUUAGGUGAAAAAG 2045 2182-2204 AD-520067.3 UUUCACCUAACUAAAAUAAUU 1912 2185-2205 AAUUAUUUUAGUUAGGUGAAAAA 2046 2183-2205 AD-75289.4 UUCACCUAACUAAAAUAAUGU 1913 2186-2206 ACAUUAUUUUAGUUAGGUGAAAA 2047 2184-2206 AD-520068.3 UCACCUAACUAAAAUAAUGUU 1914 2187-2207 AACAUUAUUUUAGUUAGGUGAAA 2048 2185-2207 AD-520069.3 CACCUAACUAAAAUAAUGUUU 1915 2188-2208 AAACAUUAUUUUAGUUAGGUGAA 2049 2186-2208 AD-520087.1 AUGUAAGGAAGCGUUGUUACU 1916 2227-2247 AGUAACAACGCUUCCUUACAUUU 2050 2225-2247 AD-520095.1 AAGCGUUGUUACCUGUUGAAU 1917 2235-2255 AUUCAACAGGUAACAACGCUUCC 2051 2233-2255 AD-520096.1 AGCGUUGUUACCUGUUGAAUU 1918 2236-2256 AAUUCAACAGGUAACAACGCUUC 2052 2234-2256 AD-520098.2 GUUGUUACCUGUUGAAUUUUU 1919 2239-2259 AAAAAUUCAACAGGUAACAACGC 2053 2237-2259 AD-75256.2 UUGUUACCUGUUGAAUUUUGU 1920 2240-2260 ACAAAAUUCAACAGGUAACAACG 2054 2238-2260 AD-520099.3 UGUUACCUGUUGAAUUUUGUU 1921 2241-2261 AACAAAAUUCAACAGGUAACAAC 2055 2239-2261 AD-67575.8 UUACCUGUUGAAUUUUGUAUU 1922 2243-2263 AAUACAAAAUUCAACAGGUAACA 2056 2241-2263 AD-520101.3 UACCUGUUGAAUUUUGUAUUU 1923 2244-2264 AAAUACAAAAUUCAACAGGUAAC 2057 2242-2264 AD-67605.8 ACCUGUUGAAUUUUGUAUUAU 1924 2245-2265 AUAAUACAAAAUUCAACAGGUAA 2058 2243-2265 AD-520102.1 CUGUUGAAUUUUGUAUUAUGU 1925 2247-2267 ACAUAAUACAAAAUUCAACAGGU 2059 2245-2267 AD-520103.1 GUUGAAUUUUGUAUUAUGUGU 1926 2249-2269 ACACAUAAUACAAAAUUCAACAG 2060 2247-2269 AD-520123.1 CAGUGAGAUGUUAGUAGAAUU 1927 2272-2292 AAUUCUACUAACAUCUCACUGAU 2061 2270-2292 AD-520127.1 GAGAUGUUAGUAGAAUAAGCU 1928 2276-2296 AGCUUAUUCUACUAACAUCUCAC 2062 2274-2296 AD-520133.1 UAGUAGAAUAAGCCUUAAAAU 1929 2283-2303 AUUUUAAGGCUUAUUCUACUAAC 2063 2281-2303 AD-520350.1 GCUGAGAUUGCACCAUUUCAU 1930 2542-2562 AUGAAAUGGUGCAAUCUCAGCUC 2064 2540-2562 AD-520351.1 CUGAGAUUGCACCAUUUCAUU 1931 2543-2563 AAUGAAAUGGUGCAAUCUCAGCU 2065 2541-2563 AD-520352.2 UGAGAUUGCACCAUUUCAUUU 1932 2544-2564 AAAUGAAAUGGUGCAAUCUCAGC 2066 2542-2564 AD-520354.1 AGAUUGCACCAUUUCAUUCCU 1933 2546-2566 AGGAAUGAAAUGGUGCAAUCUCA 2067 2544-2566 AD-520391.1 AACAAAAUAAUCUAGUGUGCU 1934 2616-2636 AGCACACUAGAUUAUUUUGUUUU 2068 2614-2636 AD-520393.1 CAAAAUAAUCUAGUGUGCAGU 1935 2618-2638 ACUGCACACUAGAUUAUUUUGUU 2069 2616-2638 AD-520467.2 UUGAACCUGGCUUAUUUUCUU 1936 2714-2734 AAGAAAAUAAGCCAGGUUCAAGU 2070 2712-2734 AD-520481.1 CACAUGGUCAGUGAGUUUCUU 1937 2748-2768 AAGAAACUCACUGACCAUGUGGG 2071 2746-2768

TABLE 33 Modified Sense and Antisense Strand Sequencesof PNPLA3 dsRNA Agents Duplex Name Sense Sequence 5′ to 3′ SEQ ID NO: Antisense Sequence 5′ to 3′ SEQ ID NO: mRNA Target Sequence SEQ ID NO: AD-518658.1 gsusccucUfcAfGfAfucuugugcguL96 2072 asCfsgcaCfaAfGfaucuGfaGfaggacscsu 2206 AGGUCCUCUCAGAUCUUGUGCGG 2340 AD-518707.1 csasuccaUfcCfUfUfcaacuuaaguL96 2073 asCfsuuaAfgUfUfgaagGfaUfggaugsgsa 2207 UCCAUCCAUCCUUCAACUUAAGC 2341 AD-518708.1 asusccauCfcUfUfCfaacuuaagcuL96 2074 asGfscuuAfaGfUfugaaGfgAfuggausgsg 2208 CCAUCCAUCCUUCAACUUAAGCA 2342 AD-518709.1 uscscaucCfuUfCfAfacuuaagcauL96 2075 asUfsgcuUfaAfGfuugaAfgGfauggasusg 2209 CAUCCAUCCUUCAACUUAAGCAA 2343 AD-518722.1 ususaagcAfaGfUfUfccuccgacauL96 2076 asUfsgucGfgAfGfgaacUfuGfcuuaasgsu 2210 ACUUAAGCAAGUUCCUCCGACAG 2344 AD-518789.1 gscsaaaaUfaGfGfCfaucucucuuuL96 2077 asAfsagaGfaGfAfugccUfaUfuuugcscsg 2211 CGGCAAAAUAGGCAUCUCUCUUA 2345 AD-518791.1 asasaauaGfgCfAfUfcucucuuacuL96 2078 asGfsuaaGfaGfAfgaugCfcUfauuuusgsc 2212 GCAAAAUAGGCAUCUCUCUUACC 2346 AD-518792.1 asasauagGfcAfUfCfucucuuaccuL96 2079 asGfsguaAfgAfGfagauGfcCfuauuususg 2213 CAAAAUAGGCAUCUCUCUUACCA 2347 AD-518794.1 asusaggcAfuCfUfCfucuuaccaguL96 2080 asCfsuggUfaAfGfagagAfuGfccuaususu 2214 AAAUAGGCAUCUCUCUUACCAGA 2348 AD-518800.1 asuscucuCfuUfAfCfcagagugucuL96 2081 asGfsacaCfuCfUfgguaAfgAfgagausgsc 2215 GCAUCUCUCUUACCAGAGUGUCU 2349 AD-518827.1 ascsuuucGfgUfCfCfaaagacgaauL96 2082 asUfsucgUfcUfUfuggaCfcGfaaaguscsa 2216 UGACUUUCGGUCCAAAGACGAAG 2350 AD-518829.1 ususucggUfcCfAfAfagacgaaguuL96 2083 asAfscuuCfgUfCfuuugGfaCfcgaaasgsu 2217 ACUUUCGGUCCAAAGACGAAGUC 2351 AD-518831.2 csgsguccAfaAfGfAfcgaagucguuL96 2084 asAfscgaCfuUfCfgucuUfuGfgaccgsasa 2218 UUCGGUCCAAAGACGAAGUCGUG 2352 AD-518832.2 gsgsuccaAfaGfAfCfgaagucguguL96 2085 asCfsacgAfcUfUfcgucUfuUfggaccsgsa 2219 UCGGUCCAAAGACGAAGUCGUGG 2353 AD-518857.1 ususgguaUfgUfUfCfcugcuucauuL96 2086 asAfsugaAfgCfAfggaaCfaUfaccaasgsg 2220 CCUUGGUAUGUUCCUGCUUCAUC 2354 AD-518921.2 usgsacaaCfgUfAfCfccuucauuguL96 2087 asCfsaauGfaAfGfgguaCfgUfugucascsu 2221 AGUGACAACGUACCCUUCAUUGA 2355 AD-518922.2 gsascaacGfuAfCfCfcuucauugauL96 2088 asUfscaaUfgAfAfggguAfcGfuugucsasc 2222 GUGACAACGUACCCUUCAUUGAU 2356 AD-518923.2 ascsaacgUfaCfCfCfuucauugauuL96 2089 asAfsucaAfuGfAfagggUfaCfguuguscsa 2223 UGACAACGUACCCUUCAUUGAUG 2357 AD-518924.2 csasacguAfcCfCfUfucauugauguL96 2090 asCfsaucAfaUfGfaaggGfuAfcguugsusc 2224 GACAACGUACCCUUCAUUGAUGC 2358 AD-518925.2 asascguaCfcCfUfUfcauugaugcuL96 2091 asGfscauCfaAfUfgaagGfgUfacguusgsu 2225 ACAACGUACCCUUCAUUGAUGCC 2359 AD-518967.1 usasaaguCfaAfGfUfccacgaacuuL96 2092 asAfsguuCfgUfGfgacuUfgAfcuuuasgsg 2226 CCUAAAGUCAAGUCCACGAACUU 2360 AD-518968.1 asasagucAfaGfUfCfcacgaacuuuL96 2093 asAfsaguUfcGfUfggacUfuGfacuuusasg 2227 CUAAAGUCAAGUCCACGAACUUU 2361 AD-518976.1 uscscacgAfaCfUfUfucuucauguuL96 2094 asAfscauGfaAfGfaaagUfuCfguggascsu 2228 AGUCCACGAACUUUCUUCAUGUG 2362 AD-518977.1 cscsacgaAfcUfUfUfcuucauguguL96 2095 asCfsacaUfgAfAfgaaaGfuUfcguggsasc 2229 GUCCACGAACUUUCUUCAUGUGG 2363 AD-518978.1 csascgaaCfuUfUfCfuucaugugguL96 2096 asCfscacAfuGfAfagaaAfgUfucgugsgsa 2230 UCCACGAACUUUCUUCAUGUGGA 2364 AD-518979.1 ascsgaacUfuUfCfUfucauguggauL96 2097 asUfsccaCfaUfGfaagaAfaGfuucgusgsg 2231 CCACGAACUUUCUUCAUGUGGAC 2365 AD-518980.2 csgsaacuUfuCfUfUfcauguggacuL96 2098 asGfsuccAfcAfUfgaagAfaAfguucgsusg 2232 CACGAACUUUCUUCAUGUGGACA 2366 AD-519043.1 ususcucuCfgAfGfAfgcuuuugucuL96 2099 asGfsacaAfaAfGfcucuCfgAfgagaasgsg 2233 CCUUCUCUCGAGAGCUUUUGUCC 2367 AD-519340.1 usgscuacCfcAfUfUfaggauaauguL96 2100 asCfsauuAfuCfCfuaauGfgGfuagcasasg 2234 CUUGCUACCCAUUAGGAUAAUGU 2368 AD-519341.1 gscsuaccCfaUfUfAfggauaauguuL96 2101 asAfscauUfaUfCfcuaaUfgGfguagcsasa 2235 UUGCUACCCAUUAGGAUAAUGUC 2369 AD-519342.1 csusacccAfuUfAfGfgauaaugucuL96 2102 asGfsacaUfuAfUfccuaAfuGfgguagscsa 2236 UGCUACCCAUUAGGAUAAUGUCU 2370 AD-519343.1 usascccaUfuAfGfGfauaaugucuuL96 2103 asAfsgacAfuUfAfuccuAfaUfggguasgsc 2237 GCUACCCAUUAGGAUAAUGUCUU 2371 AD-519344.1 ascsccauUfaGfGfAfuaaugucuuuL96 2104 asAfsagaCfaUfUfauccUfaAfugggusasg 2238 CUACCCAUUAGGAUAAUGUCUUA 2372 AD-519349.1 ususaggaUfaAfUfGfucuuauguauL96 2105 asUfsacaUfaAfGfacauUfaUfccuaasusg 2239 CAUUAGGAUAAUGUCUUAUGUAA 2373 AD-519350.1 usasggauAfaUfGfUfcuuauguaauL96 2106 asUfsuacAfuAfAfgacaUfuAfuccuasasu 2240 AUUAGGAUAAUGUCUUAUGUAAU 2374 AD-519477.1 uscsacagGfuGfUfUfcacucgaguuL96 2107 asAfscucGfaGfUfgaacAfcCfugugasgsg 2241 CCUCACAGGUGUUCACUCGAGUG 2375 AD-519596.1 csusugggCfaAfUfAfaaguaccuguL96 2108 asCfsaggUfaCfUfuuauUfgCfccaagsasa 2242 UUCUUGGGCAAUAAAGUACCUGC 2376 AD-519622.1 ususcccaGfuUfUfUfucacuagaguL96 2109 asCfsucuAfgUfGfaaaaAfcUfgggaasasg 2243 CUUUCCCAGUUUUUCACUAGAGA 2377 AD-519666.1 gsgscgagUfcUfAfGfcagauucuuuL96 2110 asAfsagaAfuCfUfgcuaGfaCfucgccsusc 2244 GAGGCGAGUCUAGCAGAUUCUUU 2378 AD-519667.1 gscsgaguCfuAfGfCfagauucuuuuL96 2111 asAfsaagAfaUfCfugcuAfgAfcucgcscsu 2245 AGGCGAGUCUAGCAGAUUCUUUC 2379 AD-519668.1 csgsagucUfaGfCfAfgauucuuucuL96 2112 asGfsaaaGfaAfUfcugcUfaGfacucgscsc 2246 GGCGAGUCUAGCAGAUUCUUUCA 2380 AD-519669.1 gsasgucuAfgCfAfGfauucuuucauL96 2113 asUfsgaaAfgAfAfucugCfuAfgacucsgsc 2247 GCGAGUCUAGCAGAUUCUUUCAG 2381 AD-519670.1 asgsucuaGfcAfGfAfuucuuucaguL96 2114 asCfsugaAfaGfAfaucuGfcUfagacuscsg 2248 CGAGUCUAGCAGAUUCUUUCAGA 2382 AD-519671.1 gsuscuagCfaGfAfUfucuuucagauL96 2115 asUfscugAfaAfGfaaucUfgCfuagacsusc 2249 GAGUCUAGCAGAUUCUUUCAGAG 2383 AD-519672.1 uscsuagcAfgAfUfUfcuuucagaguL96 2116 asCfsucuGfaAfAfgaauCfuGfcuagascsu 2250 AGUCUAGCAGAUUCUUUCAGAGG 2384 AD-519691.1 usgscuaaAfgUfUfUfcccaucuuuuL96 2117 asAfsaagAfuGfGfgaaaCfuUfuagcascsc 2251 GGUGCUAAAGUUUCCCAUCUUUG 2385 AD-519692.1 gscsuaaaGfuUfUfCfccaucuuuguL96 2118 asCfsaaaGfaUfGfggaaAfcUfuuagcsasc 2252 GUGCUAAAGUUUCCCAUCUUUGU 2386 AD-519693.1 csusaaagUfuUfCfCfcaucuuuguuL96 2119 asAfscaaAfgAfUfgggaAfaCfuuuagscsa 2253 UGCUAAAGUUUCCCAUCUUUGUG 2387 AD-519694.1 usasaaguUfuCfCfCfaucuuuguguL96 2120 asCfsacaAfaGfAfugggAfaAfcuuuasgsc 2254 GCUAAAGUUUCCCAUCUUUGUGC 2388 AD-519696.1 asasguuuCfcCfAfUfcuuugugcauL96 2121 asUfsgcaCfaAfAfgaugGfgAfaacuususa 2255 UAAAGUUUCCCAUCUUUGUGCAG 2389 AD-67554.6 uscsugagCfuGfAfGfuugguuuuauL96 2122 asUfsaaaAfcCfAfacucAfgCfucagasgsg 2256 CCUCUGAGCUGAGUUGGUUUUAU 2390 AD-519752.2 csusgagcUfgAfGfUfugguuuuauuL96 2123 asAfsuaaAfaCfCfaacuCfaGfcucagsasg 2257 CUCUGAGCUGAGUUGGUUUUAUG 2391 AD-519753.2 usgsagcuGfaGfUfUfgguuuuauguL96 2124 asCfsauaAfaAfCfcaacUfcAfgcucasgsa 2258 UCUGAGCUGAGUUGGUUUUAUGA 2392 AD-519754.8 gsasgcugAfgUfUfGfguuuuaugauL96 2125 asUfscauAfaAfAfccaaCfuCfagcucsasg 2259 CUGAGCUGAGUUGGUUUUAUGAA 2393 AD-519755.4 asgscugaGfuUfGfGfuuuuaugaauL96 2126 asUfsucaUfaAfAfaccaAfcUfcagcuscsa 2260 UGAGCUGAGUUGGUUUUAUGAAA 2394 AD-519759.2 gsasguugGfuUfUfUfaugaaaagcuL96 2127 asGfscuuUfuCfAfuaaaAfcCfaacucsasg 2261 CUGAGUUGGUUUUAUGAAAAGCU 2395 AD-519760.2 asgsuuggUfuUfUfAfugaaaagcuuL96 2128 asAfsgcuUfuUfCfauaaAfaCfcaacuscsa 2262 UGAGUUGGUUUUAUGAAAAGCUA 2396 AD-519761.2 gsusugguUfuUfAfUfgaaaagcuauL96 2129 asUfsagcUfuUfUfcauaAfaAfccaacsusc 2263 GAGUUGGUUUUAUGAAAAGCUAG 2397 AD-519766.2 ususuuauGfaAfAfAfgcuaggaaguL96 2130 asCfsuucCfuAfGfcuuuUfcAfuaaaascsc 2264 GGUUUUAUGAAAAGCUAGGAAGC 2398 AD-519773.1 asasaagcUfaGfGfAfagcaaccuuuL96 2131 asAfsaggUfuGfCfuuccUfaGfcuuuuscsa 2265 UGAAAAGCUAGGAAGCAACCUUU 2399 AD-519776.1 asgscuagGfaAfGfCfaaccuuucguL96 2132 asCfsgaaAfgGfUfugcuUfcCfuagcususu 2266 AAAGCUAGGAAGCAACCUUUCGC 2400 AD-519777.1 gscsuaggAfaGfCfAfaccuuucgcuL96 2133 asGfscgaAfaGfGfuugcUfuCfcuagcsusu 2267 AAGCUAGGAAGCAACCUUUCGCC 2401 AD-519779.1 usasggaaGfcAfAfCfcuuucgccuuL96 2134 asAfsggcGfaAfAfgguuGfcUfuccuasgsc 2268 GCUAGGAAGCAACCUUUCGCCUG 2402 AD-519809.1 cscsagcaCfuUfAfAfcucuaauacuL96 2135 asGfsuauUfaGfAfguuaAfgUfgcuggsasc 2269 GUCCAGCACUUAACUCUAAUACA 2403 AD-519810.1 csasgcacUfuAfAfCfucuaauacauL96 2136 asUfsguaUfuAfGfaguuAfaGfugcugsgsa 2270 UCCAGCACUUAACUCUAAUACAU 2404 AD-519811.1 asgscacuUfaAfCfUfcuaauacauuL96 2137 asAfsuguAfuUfAfgaguUfaAfgugcusgsg 2271 CCAGCACUUAACUCUAAUACAUC 2405 AD-519814.1 ascsuuaaCfuCfUfAfauacaucaguL96 2138 asCfsugaUfgUfAfuuagAfgUfuaagusgsc 2272 GCACUUAACUCUAAUACAUCAGC 2406 AD-519820.1 csuscuaaUfaCfAfUfcagcaugcguL96 2139 asCfsgcaUfgCfUfgaugUfaUfuagagsusu 2273 AACUCUAAUACAUCAGCAUGCGU 2407 AD-519821.1 uscsuaauAfcAfUfCfagcaugcguuL96 2140 asAfscgcAfuGfCfugauGfuAfuuagasgsu 2274 ACUCUAAUACAUCAGCAUGCGUU 2408 AD-519822.1 csusaauaCfaUfCfAfgcaugcguuuL96 2141 asAfsacgCfaUfGfcugaUfgUfauuagsasg 2275 CUCUAAUACAUCAGCAUGCGUUA 2409 AD-519823.1 usasauacAfuCfAfGfcaugcguuauL96 2142 asUfsaacGfcAfUfgcugAfuGfuauuasgsa 2276 UCUAAUACAUCAGCAUGCGUUAA 2410 AD-519826.1 usascaucAfgCfAfUfgcguuaauuuL96 2143 asAfsauuAfaCfGfcaugCfuGfauguasusu 2277 AAUACAUCAGCAUGCGUUAAUUC 2411 AD-519827.1 ascsaucaGfcAfUfGfcguuaauucuL96 2144 asGfsaauUfaAfCfgcauGfcUfgaugusasu 2278 AUACAUCAGCAUGCGUUAAUUCA 2412 AD-519828.3 csasucagCfaUfGfCfguuaauucauL96 2145 asUfsgaaUfuAfAfcgcaUfgCfugaugsusa 2279 UACAUCAGCAUGCGUUAAUUCAG 2413 AD-519830.1 uscsagcaUfgCfGfUfuaauucagcuL96 2146 asGfscugAfaUfUfaacgCfaUfgcugasusg 2280 CAUCAGCAUGCGUUAAUUCAGCU 2414 AD-519832.1 asgscaugCfgUfUfAfauucagcuguL96 2147 asCfsagcUfgAfAfuuaaCfgCfaugcusgsa 2281 UCAGCAUGCGUUAAUUCAGCUGG 2415 AD-519887.1 gsuscccuUfaCfUfGfacuguuucguL96 2148 asCfsgaaAfcAfGfucagUfaAfgggacscsc 2282 GGGUCCCUUACUGACUGUUUCGU 2416 AD-519888.1 uscsccuuAfcUfGfAfcuguuucguuL96 2149 asAfscgaAfaCfAfgucaGfuAfagggascsc 2283 GGUCCCUUACUGACUGUUUCGUG 2417 AD-519889.1 cscscuuaCfuGfAfCfuguuucguguL96 2150 asCfsacgAfaAfCfagucAfgUfaagggsasc 2284 GUCCCUUACUGACUGUUUCGUGG 2418 AD-519890.1 cscsuuacUfgAfCfUfguuucgugguL96 2151 asCfscacGfaAfAfcaguCfaGfuaaggsgsa 2285 UCCCUUACUGACUGUUUCGUGGC 2419 AD-519929.1 ususccagCfaUfGfAfgguucuuaguL96 2152 asCfsuaaGfaAfCfcucaUfgCfuggaascsa 2286 UGUUCCAGCAUGAGGUUCUUAGA 2420 AD-519931.1 cscsagcaUfgAfGfGfuucuuagaauL96 2153 asUfsucuAfaGfAfaccuCfaUfgcuggsasa 2287 UUCCAGCAUGAGGUUCUUAGAAU 2421 AD-519932.1 csasgcauGfaGfGfUfucuuagaauuL96 2154 asAfsuucUfaAfGfaaccUfcAfugcugsgsa 2288 UCCAGCAUGAGGUUCUUAGAAUG 2422 AD-519933.1 asgscaugAfgGfUfUfcuuagaauguL96 2155 asCfsauuCfuAfAfgaacCfuCfaugcusgsg 2289 CCAGCAUGAGGUUCUUAGAAUGA 2423 AD-519935.2 csasugagGfuUfCfUfuagaaugacuL96 2156 asGfsucaUfuCfUfaagaAfcCfucaugscsu 2290 AGCAUGAGGUUCUUAGAAUGACA 2424 AD-519942.1 ususcuuaGfaAfUfGfacagguguuuL96 2157 asAfsacaCfcUfGfucauUfcUfaagaascsc 2291 GGUUCUUAGAAUGACAGGUGUUU 2425 AD-520005.1 gsgsucugCfaAfAfGfaugauaaccuL96 2158 asGfsguuAfuCfAfucuuUfgCfagaccsasc 2292 GUGGUCUGCAAAGAUGAUAACCU 2426 AD-520006.1 gsuscugcAfaAfGfAfugauaaccuuL96 2159 asAfsgguUfaUfCfaucuUfuGfcagacscsa 2293 UGGUCUGCAAAGAUGAUAACCUU 2427 AD-520007.1 uscsugcaAfaGfAfUfgauaaccuuuL96 2160 asAfsaggUfuAfUfcaucUfuUfgcagascsc 2294 GGUCUGCAAAGAUGAUAACCUUG 2428 AD-520008.1 csusgcaaAfgAfUfGfauaaccuuguL96 2161 asCfsaagGfuUfAfucauCfuUfugcagsasc 2295 GUCUGCAAAGAUGAUAACCUUGA 2429 AD-520011.1 csasaagaUfgAfUfAfaccuugacuuL96 2162 asAfsgucAfaGfGfuuauCfaUfcuuugscsa 2296 UGCAAAGAUGAUAACCUUGACUA 2430 AD-520020.1 usasaccuUfgAfCfUfacuaaaaacuL96 2163 asGfsuuuUfuAfGfuaguCfaAfgguuasusc 2297 GAUAACCUUGACUACUAAAAACG 2431 AD-520021.1 asasccuuGfaCfUfAfcuaaaaacguL96 2164 asCfsguuUfuUfAfguagUfcAfagguusasu 2298 AUAACCUUGACUACUAAAAACGU 2432 AD-520022.1 ascscuugAfcUfAfCfuaaaaacguuL96 2165 asAfscguUfuUfUfaguaGfuCfaaggususa 2299 UAACCUUGACUACUAAAAACGUC 2433 AD-520023.1 cscsuugaCfuAfCfUfaaaaacgucuL96 2166 asGfsacgUfuUfUfuaguAfgUfcaaggsusu 2300 AACCUUGACUACUAAAAACGUCU 2434 AD-520024.1 csusugacUfaCfUfAfaaaacgucuuL96 2167 asAfsgacGfuUfUfuuagUfaGfucaagsgsu 2301 ACCUUGACUACUAAAAACGUCUC 2435 AD-520025.1 ususgacuAfcUfAfAfaaacgucucuL96 2168 asGfsagaCfgUfUfuuuaGfuAfgucaasgsg 2302 CCUUGACUACUAAAAACGUCUCC 2436 AD-520034.1 gsgsguaaCfaAfGfAfugauaaucuuL96 2169 asAfsgauUfaUfCfaucuUfgUfuacccscsc 2303 GGGGGUAACAAGAUGAUAAUCUA 2437 AD-520035.3 gsgsuaacAfaGfAfUfgauaaucuauL96 2170 asUfsagaUfuAfUfcaucUfuGfuuaccscsc 2304 GGGGUAACAAGAUGAUAAUCUAC 2438 AD-520036.1 gsusaacaAfgAfUfGfauaaucuacuL96 2171 asGfsuagAfuUfAfucauCfuUfguuacscsc 2305 GGGUAACAAGAUGAUAAUCUACU 2439 AD-520037.1 usasacaaGfaUfGfAfuaaucuacuuL96 2172 asAfsguaGfaUfUfaucaUfcUfuguuascsc 2306 GGUAACAAGAUGAUAAUCUACUU 2440 AD-520038.1 asascaagAfuGfAfUfaaucuacuuuL96 2173 asAfsaguAfgAfUfuaucAfuCfuuguusasc 2307 GUAACAAGAUGAUAAUCUACUUA 2441 AD-520053.1 ususuuagAfaCfAfCfcuuuuucacuL96 2174 asGfsugaAfaAfAfggugUfuCfuaaaasusu 2308 AAUUUUAGAACACCUUUUUCACC 2442 AD-520060.3 ascsaccuUfuUfUfCfaccuaacuauL96 2175 asUfsaguUfaGfGfugaaAfaAfggugususc 2309 GAACACCUUUUUCACCUAACUAA 2443 AD-520061.3 csasccuuUfuUfCfAfccuaacuaauL96 2176 asUfsuagUfuAfGfgugaAfaAfaggugsusu 2310 AACACCUUUUUCACCUAACUAAA 2444 AD-520064.5 csusuuuuCfaCfCfUfaacuaaaauuL96 2177 asAfsuuuUfaGfUfuaggUfgAfaaaagsgsu 2311 ACCUUUUUCACCUAACUAAAAUA 2445 AD-520065.4 ususuuucAfcCfUfAfacuaaaauauL96 2178 asUfsauuUfuAfGfuuagGfuGfaaaaasgsg 2312 CCUUUUUCACCUAACUAAAAUAA 2446 AD-520066.2 ususuucaCfcUfAfAfcuaaaauaauL96 2179 asUfsuauUfuUfAfguuaGfgUfgaaaasasg 2313 CUUUUUCACCUAACUAAAAUAAU 2447 AD-520067.3 ususucacCfuAfAfCfuaaaauaauuL96 2180 asAfsuuaUfuUfUfaguuAfgGfugaaasasa 2314 UUUUUCACCUAACUAAAAUAAUG 2448 AD-75289.4 ususcaccUfaAfCfUfaaaauaauguL96 2181 asCfsauuAfuUfUfuaguUfaGfgugaasasa 2315 UUUUCACCUAACUAAAAUAAUGU 2449 AD-520068.3 uscsaccuAfaCfUfAfaaauaauguuL96 2182 asAfscauUfaUfUfuuagUfuAfggugasasa 2316 UUUCACCUAACUAAAAUAAUGUU 2450 AD-520069.3 csasccuaAfcUfAfAfaauaauguuuL96 2183 asAfsacaUfuAfUfuuuaGfuUfaggugsasa 2317 UUCACCUAACUAAAAUAAUGUUU 2451 AD-520087.1 asusguaaGfgAfAfGfcguuguuacuL96 2184 asGfsuaaCfaAfCfgcuuCfcUfuacaususu 2318 AAAUGUAAGGAAGCGUUGUUACC 2452 AD-520095.1 asasgcguUfgUfUfAfccuguugaauL96 2185 asUfsucaAfcAfGfguaaCfaAfcgcuuscsc 2319 GGAAGCGUUGUUACCUGUUGAAU 2453 AD-520096.1 asgscguuGfuUfAfCfcuguugaauuL96 2186 asAfsuucAfaCfAfgguaAfcAfacgcususc 2320 GAAGCGUUGUUACCUGUUGAAUU 2454 AD-520098.2 gsusuguuAfcCfUfGfuugaauuuuuL96 2187 asAfsaaaUfuCfAfacagGfuAfacaacsgsc 2321 GCGUUGUUACCUGUUGAAUUUUG 2455 AD-75256.2 ususguuaCfcUfGfUfugaauuuuguL96 2188 asCfsaaaAfuUfCfaacaGfgUfaacaascsg 2322 CGUUGUUACCUGUUGAAUUUUGU 2456 AD-520099.3 usgsuuacCfuGfUfUfgaauuuuguuL96 2189 asAfscaaAfaUfUfcaacAfgGfuaacasasc 2323 GUUGUUACCUGUUGAAUUUUGUA 2457 AD-67575.8 ususaccuGfuUfGfAfauuuuguauuL96 2190 asAfsuacAfaAfAfuucaAfcAfgguaascsa 2324 UGUUACCUGUUGAAUUUUGUAUU 2458 AD-520101.3 usasccugUfuGfAfAfuuuuguauuuL96 2191 asAfsauaCfaAfAfauucAfaCfagguasasc 2325 GUUACCUGUUGAAUUUUGUAUUA 2459 AD-67605.8 ascscuguUfgAfAfUfuuuguauuauL96 2192 asUfsaauAfcAfAfaauuCfaAfcaggusasa 2326 UUACCUGUUGAAUUUUGUAUUAU 2460 AD-520102.1 csusguugAfaUfUfUfuguauuauguL96 2193 asCfsauaAfuAfCfaaaaUfuCfaacagsgsu 2327 ACCUGUUGAAUUUUGUAUUAUGU 2461 AD-520103.1 gsusugaaUfuUfUfGfuauuauguguL96 2194 asCfsacaUfaAfUfacaaAfaUfucaacsasg 2328 CUGUUGAAUUUUGUAUUAUGUGA 2462 AD-520123.1 csasgugaGfaUfGfUfuaguagaauuL96 2195 asAfsuucUfaCfUfaacaUfcUfcacugsasu 2329 AUCAGUGAGAUGUUAGUAGAAUA 2463 AD-520127.1 gsasgaugUfuAfGfUfagaauaagcuL96 2196 asGfscuuAfuUfCfuacuAfaCfaucucsasc 2330 GUGAGAUGUUAGUAGAAUAAGCC 2464 AD-520133.1 usasguagAfaUfAfAfgccuuaaaauL96 2197 asUfsuuuAfaGfGfcuuaUfuCfuacuasasc 2331 GUUAGUAGAAUAAGCCUUAAAAA 2465 AD-520350.1 gscsugagAfuUfGfCfaccauuucauL96 2198 asUfsgaaAfuGfGfugcaAfuCfucagcsusc 2332 GAGCUGAGAUUGCACCAUUUCAU 2466 AD-520351.1 csusgagaUfuGfCfAfccauuucauuL96 2199 asAfsugaAfaUfGfgugcAfaUfcucagscsu 2333 AGCUGAGAUUGCACCAUUUCAUU 2467 AD-520352.2 usgsagauUfgCfAfCfcauuucauuuL96 2200 asAfsaugAfaAfUfggugCfaAfucucasgsc 2334 GCUGAGAUUGCACCAUUUCAUUC 2468 AD-520354.1 asgsauugCfaCfCfAfuuucauuccuL96 2201 asGfsgaaUfgAfAfauggUfgCfaaucuscsa 2335 UGAGAUUGCACCAUUUCAUUCCA 2469 AD-520391.1 asascaaaAfuAfAfUfcuagugugcuL96 2202 asGfscacAfcUfAfgauuAfuUfuuguususu 2336 AAAACAAAAUAAUCUAGUGUGCA 2470 AD-520393.1 csasaaauAfaUfCfUfagugugcaguL96 2203 asCfsugcAfcAfCfuagaUfuAfuuuugsusu 2337 AACAAAAUAAUCUAGUGUGCAGG 2471 AD-520467.2 ususgaacCfuGfGfCfuuauuuucuuL96 2204 asAfsgaaAfaUfAfagccAfgGfuucaasgsu 2338 ACUUGAACCUGGCUUAUUUUCUG 2472 AD-520481.1 csascaugGfuCfAfGfugaguuucuuL96 2205 asAfsgaaAfcUfCfacugAfcCfaugugsgsg 2339 CCCACAUGGUCAGUGAGUUUCUC 2473

TABLE 34 PNPLA3 Single Dose Screen in Hep3B Cells 50 nM 10 nM 1 nM 0.1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-518658.1 42.94766 12.41792 64.82536 23.85209 100.7428 19.40937 93.04989 10.43198 AD-518707.1 55.62284 10.27535 71.93996 7.142065 105.6313 6.809517 101.7458 24.26104 AD-518708.1 69.75236 17.4307 65.43961 13.43465 116.9685 10.62334 108.8084 20.02657 AD-518709.1 50.31404 5.652726 47.3224 5.147422 84.40248 16.07951 103.3354 20.86118 AD-518722.1 69.92801 9.598491 50.6688 17.8235 95.22432 19.21756 96.11806 12.44369 AD-518789.1 32.53758 5.593675 32.77075 4.054891 50.71233 7.306018 65.33746 3.716173 AD-518791.1 33.37483 4.059424 42.05998 3.447412 84.64916 8.215959 78.26863 11.97385 AD-518792.1 43.20357 13.54477 55.52443 3.322024 87.85942 9.678187 75.64313 15.3978 AD-518794.1 48.32774 13.80689 73.9973 15.10836 95.08435 15.93579 94.333 19.92082 AD-518800.1 69.62243 3.754956 78.00498 8.905591 126.5248 12.07819 87.10116 4.495733 AD-518827.1 66.84976 1.87091 64.05565 7.824748 109.3026 15.19898 122.0295 26.52146 AD-518829.1 95.0517 11.75578 91.47261 5.656564 127.3295 25.96408 123.1953 22.12334 AD-518831.2 54.44406 8.796633 55.56703 8.592537 75.10855 12.05533 94.42534 11.73385 AD-518832.2 72.24416 13.66241 62.79827 10.55016 88.30892 15.80649 99.79269 13.66623 AD-518857.1 47.26442 8.72365 39.35137 6.083911 68.1639 8.1472 85.29716 11.93514 AD-518921.2 67.47179 23.73035 66.31535 9.437307 96.34766 15.71656 71.63637 13.82891 AD-518922.2 42.15927 11.13492 54.55048 17.92362 86.58956 13.01162 98.89853 6.354892 AD-518923.2 41.7079 3.949238 46.7206 9.026298 58.04384 5.899113 70.61148 5.844163 AD-518924.2 59.32409 12.35522 66.42772 17.2608 93.49551 4.358715 101.9138 20.19513 AD-518925.2 80.49793 11.48081 79.82113 8.071919 85.65246 9.87835 107.6037 11.50751 AD-518967.1 65.01747 7.983442 60.89236 9.690421 68.56459 5.707428 96.17366 15.85213 AD-518968.1 61.42989 4.832082 63.15834 7.35798 71.61803 10.4465 102.5449 13.47582 AD-518976.1 86.43122 10.59274 99.15413 7.861639 106.5852 27.62984 94.79483 4.2749 AD-518977.1 97.00068 21.92919 78.96241 17.53663 92.04531 9.774873 76.36653 9.779583 AD-518978.1 52.37278 11.64162 93.20182 8.322116 108.6372 2.25339 105.309 14.97478 AD-518979.1 66.15803 16.38301 83.53397 26.14738 115.7627 17.24141 91.00506 20.14569 AD-518980.2 102.8949 13.60259 114.919 22.75057 101.7028 16.93855 137.2896 13.75517 AD-519043.1 52.27022 1.565815 65.30566 6.985576 99.69383 10.65792 118.6239 13.86267 AD-519340.1 50.02449 3.164387 59.53594 7.087385 83.13269 17.55595 104.4721 8.587117 AD-519341.1 56.5467 4.790482 71.17177 4.959802 89.62357 8.905254 106.1918 6.972196 AD-519342.1 40.34185 2.272544 51.3861 5.876967 80.09125 14.52679 95.36078 12.45925 AD-519343.1 25.54698 3.00203 27.78009 9.325246 44.82058 9.65901 84.70717 12.42313 AD-519344.1 30.81974 6.324083 42.91242 10.1455 63.99386 13.26317 70.11595 5.616455 AD-519349.1 25.64301 4.685127 35.13943 10.39843 49.6449 7.724092 63.0249 16.56877 AD-519350.1 25.37437 5.106213 33.42453 7.475218 48.36196 8.196347 77.78587 9.513747 AD-519477.1 55.15233 1.53307 58.82498 1.176072 86.54209 11.23175 129.8929 22.37189 AD-519596.1 49.32799 7.794994 66.34927 8.449974 85.59407 10.75557 122.6109 21.977 AD-519622.1 72.31278 7.77303 83.80396 3.236319 89.72191 20.42891 101.6253 16.2137 AD-519666.1 58.99223 9.174687 60.92266 2.279997 62.24535 8.846176 84.60831 9.404301 AD-519667.1 39.03083 10.13723 32.13225 3.182724 54.28087 9.091912 63.60743 16.76734 AD-519668.1 39.28227 7.783368 52.52746 15.97764 90.38161 8.507587 83.20261 10.42693 AD-519669.1 56.79735 2.773505 62.00964 8.54995 91.87774 16.31173 102.7233 30.30425 AD-519670.1 63.18069 13.12076 66.46455 5.746918 74.18941 25.33623 115.1692 6.864002 AD-519671.1 62.08825 6.619747 77.23297 14.70727 76.38787 12.4203 107.8572 12.53719 AD-519672.1 74.15201 10.12734 91.90442 7.785918 94.94023 10.03551 117.9209 5.07782 AD-519691.1 45.7187 10.19413 61.86656 16.24762 81.99914 12.03996 85.72808 10.46489 AD-519692.1 69.49335 11.27899 75.27569 10.25374 110.613 15.88181 105.2715 10.10224 AD-519693.1 54.32562 9.364318 65.18126 12.43703 77.75208 7.341201 114.9356 19.24612 AD-519694.1 56.31428 7.487224 63.04836 4.47575 79.4272 19.21656 105.4726 30.78601 AD-519696.1 80.14073 4.963576 90.4529 7.234866 106.7291 12.33685 116.2394 17.5721 AD-67554.6 32.5344 2.785433 41.83857 6.041739 39.86673 4.460883 75.97448 27.32283 AD-519752.2 43.34072 3.127806 51.03679 8.917442 46.87752 5.764831 80.92584 12.17422 AD-519753.2 25.96199 5.067472 37.45149 3.147941 48.76312 12.1361 55.46163 9.983352 AD-519754.8 50.72996 13.31669 61.07876 5.56202 89.88631 16.05322 109.8062 23.17114 AD-519755.4 51.58956 5.473844 52.51098 4.438733 63.18747 6.566833 81.27018 14.70514 AD-519759.2 63.65028 3.967476 73.12785 11.96064 102.2376 7.185976 116.2375 25.02045 AD-519760.2 62.00693 15.41249 51.28109 5.820685 69.81737 16.40661 82.37727 14.72109 AD-519761.2 53.73688 3.887523 57.02822 9.22889 64.59723 7.706789 105.7042 11.06913 AD-519766.2 50.21221 9.519207 66.10495 5.487173 88.64545 15.71198 106.0675 7.998775 AD-519773.1 29.88908 3.299812 34.55844 5.789048 44.35859 3.297106 75.38487 19.86745 AD-519776.1 33.56361 9.147108 44.87364 11.43685 62.63161 7.745073 75.53641 27.11615 AD-519777.1 58.78548 20.27898 72.5594 11.24378 106.9036 12.15886 121.0682 15.67886 AD-519779.1 60.65973 4.346633 68.90639 5.485959 96.38133 14.77649 99.24701 11.64262 AD-519809.1 51.34968 4.70846 62.59669 11.5038 85.99883 10.76169 117.9819 28.94899 AD-519810.1 52.77966 4.812098 58.85969 11.53462 75.04217 15.48752 96.04315 9.024712 AD-519811.1 41.83074 5.570013 62.54468 6.636095 68.85843 4.515473 96.25603 13.47886 AD-519814.1 43.6037 4.666228 51.40583 5.184036 70.30465 6.649075 112.0615 7.600643 AD-519820.1 35.46836 5.787243 44.97954 3.359216 75.81738 12.67708 83.99358 7.381867 AD-519821.1 30.70277 3.62287 39.00705 5.193448 71.72964 5.90817 52.42374 2.717107 AD-519822.1 24.09065 3.392701 42.99502 11.23734 79.03643 19.71305 101.1889 15.16909 AD-519823.1 53.12406 7.898687 54.03719 10.43083 85.09653 22.19466 101.8818 16.35143 AD-519826.1 42.85731 2.286875 51.97531 10.79151 81.25133 8.59057 104.7209 9.680307 AD-519827.1 79.38389 3.13229 88.43322 15.29789 103.171 8.051649 97.46346 10.30878 AD-519828.3 37.66239 5.17373 52.17304 6.709216 64.23609 3.999677 99.78564 16.62182 AD-519830.1 47.12815 8.031646 59.14945 9.487784 82.00889 18.98362 89.30526 6.13986 AD-519832.1 34.91341 4.749048 55.74108 3.769992 71.52871 4.738435 89.7728 8.801833 AD-519887.1 32.15966 9.58332 43.38057 8.765226 60.82413 30.07213 72.79899 10.84883 AD-519888.1 54.53395 11.20725 71.71064 13.2849 120.5159 24.54163 105.1235 2.614384 AD-519889.1 57.80795 12.25515 66.40043 6.480154 114.7472 10.06082 111.2871 10.37692 AD-519890.1 71.77843 5.504303 89.06564 9.311918 114.8869 22.11956 122.6381 21.95622 AD-519929.1 51.24411 4.482367 57.14287 10.9567 83.69919 13.52289 93.01579 7.833971 AD-519931.1 50.30644 3.56686 62.02151 3.375044 83.31024 5.457631 102.6883 5.397906 AD-519932.1 31.44614 11.99819 45.59588 11.73238 67.6784 21.54268 102.4539 36.79452 AD-519933.1 32.84923 3.175817 50.03872 9.146825 72.2142 5.834927 69.56286 13.82901 AD-519935.2 23.19916 5.459604 34.57125 4.267756 52.33182 7.461964 52.51531 2.774906 AD-519942.1 33.43069 7.462724 53.47246 9.257367 90.53541 13.39055 99.71394 8.678145 AD-520005.1 54.87907 8.873501 72.14087 12.56384 111.6164 14.67787 106.2691 14.61877 AD-520006.1 24.04719 2.376527 39.32581 3.470195 76.83414 16.33539 98.31059 15.6044 AD-520007.1 16.0072 2.896706 28.52854 4.013988 56.35011 5.720959 71.24665 3.904088 AD-520008.1 70.54243 7.357645 61.40247 10.0634 76.12696 10.8282 81.73514 12.70165 AD-520011.1 61.34677 18.83765 55.76087 8.165514 81.57725 16.00681 75.3319 5.415098 AD-520020.1 56.73076 8.466195 53.84936 9.58588 85.81525 20.35519 70.27459 15.07749 AD-520021.1 58.05942 7.510224 49.3573 6.039504 82.04944 15.89054 85.39012 20.28745 AD-520022.1 60.33364 4.301973 45.63061 6.280662 81.19155 15.95622 85.09768 18.42796 AD-520023.1 53.1275 5.840407 51.07057 10.97167 82.54809 4.396742 80.65711 10.21667 AD-520024.1 37.0917 8.452936 38.18884 6.75889 59.22727 13.7917 66.24876 10.41801 AD-520025.1 36.99918 5.011139 41.66326 4.435679 55.22974 9.676515 61.5233 10.29043 AD-520034.1 50.74447 16.90927 55.13512 15.82911 69.34809 9.6233 68.24787 8.182789 AD-520035.3 47.68497 13.2744 41.72851 3.402547 79.78654 18.57665 66.67162 8.660544 AD-520036.1 62.46816 8.270828 50.74637 8.326201 76.46313 11.95967 92.93795 9.492514 AD-520037.1 56.5259 8.816028 50.21156 9.119045 81.8312 14.2877 85.90267 13.16231 AD-520038.1 51.75385 18.57013 34.06313 8.261207 56.86823 7.901725 81.77106 3.523512 AD-520053.1 41.77559 8.867003 41.62096 19.77457 63.14894 8.57688 65.0597 2.993574 AD-520060.3 44.68219 8.424713 37.26257 7.035423 59.53055 7.908942 79.85002 27.59914 AD-520061.3 28.67808 4.381034 33.96453 1.79867 65.35527 12.69704 52.54273 8.841387 AD-520064.5 48.04944 8.872461 44.13648 11.70039 71.24889 12.89441 83.53806 16.61364 AD-520065.4 66.23067 11.50931 54.42123 9.981868 65.499 6.582631 95.33679 24.57588 AD-520066.2 95.65312 47.68023 67.23665 3.849851 79.22817 13.4242 92.7217 32.33753 AD-520067.3 105.1375 37.57514 43.67235 7.126124 52.75892 4.102313 101.1133 15.14941 AD-75289.4 56.22618 10.19969 62.18789 16.47643 68.09052 3.20629 96.80374 10.91438 AD-520068.3 70.91495 2.704445 53.84268 7.744443 78.77375 10.06 93.78921 4.586384 AD-520069.3 39.78116 11.92992 49.98416 7.246082 73.49029 10.7709 72.88815 20.82402 AD-520087.1 68.98191 8.459896 76.07173 15.31797 104.4457 13.22462 113.8743 43.9017 AD-520095.1 81.69757 9.807112 82.56076 31.28487 96.63698 22.73994 86.29611 9.239682 AD-520096.1 64.90297 1.936481 73.68988 26.59006 77.92236 9.635013 96.03301 15.61368 AD-520098.2 112.056 49.26435 72.75627 13.56107 71.41998 14.01052 71.59986 6.379803 AD-75256.2 75.79616 8.737346 74.33521 23.01815 85.34296 16.72191 98.87664 15.70082 AD-520099.3 59.03481 13.43313 48.04655 4.361352 65.41177 10.4964 106.4857 12.07129 AD-67575.8 35.92082 6.119518 52.70581 10.20549 62.67975 14.50294 70.73624 4.135402 AD-520101.3 58.99073 10.67199 73.28896 30.81621 93.80172 7.608287 92.6562 21.29585 AD-67605.8 59.32836 23.51253 62.08382 10.45108 90.11555 23.85401 81.39889 14.06351 AD-520102.1 75.87013 7.619236 96.86606 25.47729 86.92724 3.927143 93.45379 4.342059 AD-520103.1 82.82953 7.726372 89.38753 17.13548 98.84202 10.39095 97.07032 15.12284 AD-520123.1 102.8972 19.3969 74.9593 9.436158 77.04401 6.792248 92.67994 8.25485 AD-520127.1 73.10119 23.1491 97.15216 30.89856 91.29938 7.718378 105.2282 19.25022 AD-520133.1 36.1541 6.391883 67.15863 24.0854 62.06326 14.57401 69.3185 9.301314 AD-520350.1 58.87999 14.59136 90.84397 18.68087 80.50092 1.862191 82.76945 25.04629 AD-520351.1 71.26303 7.088025 76.54699 6.320899 137.9383 34.59137 97.85447 11.56826 AD-520352.2 85.42707 13.84644 90.42389 8.580908 107.6216 16.56475 107.883 10.17883 AD-520354.1 95.72204 19.82589 76.68991 4.160213 82.13668 14.73163 129.7049 24.2914 AD-520391.1 129.611 19.84147 123.2649 30.64217 84.44844 16.43249 104.8518 6.691812 AD-520393.1 102.3696 22.0114 108.367 33.90646 82.13201 9.440079 108.4258 13.13794 AD-520467.2 57.00049 5.905218 65.50329 11.03502 58.17843 10.07205 93.36498 19.27898 AD-520481.1 69.55206 1.607017 75.37835 8.657669 70.73405 11.67826 98.46396 27.39758

TABLE 35 PNPLA3 Single Dose Screen in Primary Cynomolgus Hepatocytes (PCH) 50 nM 10 nM 1 nM 0.1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-518658.1 42.94766 12.41792 107.2499 25.92054 108.4212 13.09521 111.2395 18.49003 AD-518707.1 55.62284 10.27535 65.31688 10.74275 87.51894 4.680768 97.55652 8.789667 AD-518708.1 69.75236 17.4307 82.07462 7.024724 101.0641 5.648431 102.4511 6.754587 AD-518709.1 50.31404 5.652726 31.56035 2.229946 53.01344 6.566759 90.32525 6.88911 AD-518722.1 69.92801 9.598491 73.56284 23.22718 87.72646 10.60739 99.94279 0.399497 AD-518789.1 32.53758 5.593675 99.3493 5.934069 108.6534 5.010722 102.1127 7.124654 AD-518791.1 33.37483 4.059424 108.3646 7.1364 106.7647 10.988 104.953 9.843194 AD-518792.1 43.20357 13.54477 102.7673 4.006107 113.5466 19.39515 98.04121 5.026568 AD-518794.1 48.32774 13.80689 111.6904 1.606519 95.31782 12.7478 119.9721 18.7764 AD-518800.1 69.62243 3.754956 102.9294 3.404207 100.6042 1.31281 105.7795 4.662372 AD-518827.1 66.84976 1.87091 43.72212 6.04683 62.32772 5.541399 89.35777 1.566393 AD-518829.1 95.0517 11.75578 46.53891 4.776352 92.57407 12.66442 97.58275 6.311261 AD-518831.2 54.44406 8.796633 33.53257 6.622253 48.34299 3.453772 77.46457 4.24481 AD-518832.2 72.24416 13.66241 44.97781 8.475713 76.43563 14.96934 94.37157 4.830518 AD-518857.1 47.26442 8.72365 25.91835 5.262805 30.45906 3.310539 50.39355 2.457611 AD-518921.2 67.47179 23.73035 38.22451 2.010726 84.3703 3.972883 111.7785 8.371431 AD-518922.2 42.15927 11.13492 25.87297 4.283199 35.38645 8.004708 65.68091 2.858227 AD-518923.2 41.7079 3.949238 18.61375 2.565515 27.95204 7.348621 45.49059 5.685225 AD-518924.2 59.32409 12.35522 27.651 2.401131 50.48284 5.945514 93.43941 8.653832 AD-518925.2 80.49793 11.48081 63.69099 8.749987 89.37984 5.319914 108.7819 17.40859 AD-518967.1 65.01747 7.983442 28.57555 2.488417 45.79737 11.52997 79.26503 14.64462 AD-518968.1 61.42989 4.832082 64.48533 11.41152 58.35085 12.27641 79.24612 4.769727 AD-518976.1 86.43122 10.59274 104.7096 7.723962 117.0872 10.04325 104.5054 11.24525 AD-518977.1 97.00068 21.92919 102.0546 8.089759 112.7422 10.42651 114.2569 13.11956 AD-518978.1 52.37278 11.64162 52.48526 2.654783 90.83673 8.68093 107.3822 14.933 AD-518979.1 66.15803 16.38301 43.33509 7.856349 73.18861 5.884268 111.2535 30.84103 AD-518980.2 102.8949 13.60259 74.721 9.340619 102.8776 15.689 101.1656 11.37242 AD-519043.1 52.27022 1.565815 79.56647 13.03313 90.18324 13.00075 111.8124 14.07459 AD-519340.1 50.02449 3.164387 33.95453 5.624572 51.64516 4.006857 89.47256 19.92119 AD-519341.1 56.5467 4.790482 33.82173 6.439423 49.92403 4.47156 79.71827 2.365924 AD-519342.1 40.34185 2.272544 22.97385 2.567424 47.15934 7.164376 82.837 10.23602 AD-519343.1 25.54698 3.00203 16.9417 2.565533 33.18726 10.34591 61.99484 12.6374 AD-519344.1 30.81974 6.324083 16.59713 2.805304 30.37118 9.688215 47.47176 16.77751 AD-519349.1 25.64301 4.685127 24.63817 2.5165 48.54704 6.286199 70.48421 10.79533 AD-519350.1 25.37437 5.106213 27.95723 4.745196 35.9648 12.83972 53.34265 8.70051 AD-519477.1 55.15233 1.53307 103.364 5.785044 105.2844 8.074213 100.4023 3.873633 AD-519596.1 49.32799 7.794994 43.34728 6.503345 85.37786 18.4169 95.97131 9.683516 AD-519622.1 72.31278 7.77303 76.02368 3.15011 94.8204 6.132603 102.1213 2.335471 AD-519666.1 58.99223 9.174687 39.62579 7.978804 41.40143 7.590112 67.90315 15.49473 AD-519667.1 39.03083 10.13723 31.64615 5.696646 33.87847 8.438246 52.31223 5.003076 AD-519668.1 39.28227 7.783368 21.24655 5.859197 29.49321 3.96825 46.35034 7.128104 AD-519669.1 56.79735 2.773505 28.77363 5.317268 34.71547 3.827615 69.77341 9.29004 AD-519670.1 63.18069 13.12076 29.26312 4.476751 41.51635 8.636287 68.66003 7.79563 AD-519671.1 62.08825 6.619747 31.30349 6.066918 43.73392 3.452455 99.40035 13.8884 AD-519672.1 74.15201 10.12734 58.05233 10.35856 76.76989 0.667929 96.65461 3.977393 AD-519691.1 45.7187 10.19413 46.15352 17.94174 51.34247 11.97931 72.33644 1.687884 AD-519692.1 69.49335 11.27899 58.99249 11.32724 71.02053 4.942421 101.379 17.91804 AD-519693.1 54.32562 9.364318 30.0908 3.788583 44.37419 9.335986 80.6514 11.02383 AD-519694.1 56.31428 7.487224 30.98432 2.54551 63.19903 12.35092 96.9065 15.10197 AD-519696.1 80.14073 4.963576 43.18088 8.202438 64.84407 18.69629 83.5461 2.868943 AD-67554.6 32.5344 2.785433 22.51723 5.938662 31.29634 6.557666 44.04866 5.20049 AD-519752.2 43.34072 3.127806 29.97457 6.32733 41.24786 11.31367 60.23414 9.95955 AD-519753.2 25.96199 5.067472 39.32706 25.6105 38.48882 8.751697 64.91725 5.699831 AD-519754.8 50.72996 13.31669 27.21654 1.411721 54.10386 10.6625 71.83422 7.262762 AD-519755.4 51.58956 5.473844 29.96824 9.177983 28.18829 2.586248 52.79346 6.606697 AD-519759.2 63.65028 3.967476 29.80889 10.47159 56.6665 3.437445 86.08391 12.83687 AD-519760.2 62.00693 15.41249 27.56752 3.854633 32.43736 4.198986 55.89127 7.165979 AD-519761.2 53.73688 3.887523 28.45853 4.818403 34.3774 1.450402 62.30843 17.80523 AD-519766.2 50.21221 9.519207 36.33141 4.453995 58.6345 8.810374 83.37992 5.951691 AD-519773.1 29.88908 3.299812 30.49178 2.190343 34.55642 5.012424 54.0685 3.92445 AD-519776.1 33.56361 9.147108 63.08933 15.8987 66.12198 6.090349 88.88788 0.928982 AD-519777.1 58.78548 20.27898 74.51794 17.44182 97.80863 5.93003 98.35458 10.33761 AD-519779.1 60.65973 4.346633 93.14756 16.52411 91.62632 14.29083 92.46408 5.672111 AD-519809.1 51.34968 4.70846 31.02454 4.751388 44.22855 9.42411 69.09952 9.938716 AD-519810.1 52.77966 4.812098 37.18433 11.01069 50.95422 11.30566 76.54755 17.24326 AD-519811.1 41.83074 5.570013 55.95473 19.52748 52.55232 4.130694 79.87171 11.5627 AD-519814.1 43.6037 4.666228 80.65601 11.97305 87.82249 9.563659 90.47957 3.164596 AD-519820.1 35.46836 5.787243 49.55763 10.92159 66.90424 11.27349 80.7228 6.515261 AD-519821.1 30.70277 3.62287 61.38392 4.112118 86.7722 7.718429 89.74907 4.664017 AD-519822.1 24.09065 3.392701 27.8375 11.24935 45.27112 14.85539 62.86481 9.88856 AD-519823.1 53.12406 7.898687 40.64473 18.3133 45.90214 13.72145 71.43003 18.06916 AD-519826.1 42.85731 2.286875 47.85482 5.871376 57.48068 5.578858 83.69736 3.162118 AD-519827.1 79.38389 3.13229 40.34436 4.544035 84.1747 8.546728 91.28862 3.911967 AD-519828.3 37.66239 5.17373 43.15043 3.723005 56.13689 4.544683 78.29178 4.68741 AD-519830.1 47.12815 8.031646 48.07977 17.07435 61.61371 6.12148 90.1255 12.71259 AD-519832.1 34.91341 4.749048 64.86568 17.49753 68.30678 3.626538 91.33426 13.59467 AD-519887.1 32.15966 9.58332 65.48012 7.894547 72.84913 4.3847 92.60784 5.197678 AD-519888.1 54.53395 11.20725 31.50922 3.110488 91.69969 3.187794 103.071 9.793602 AD-519889.1 57.80795 12.25515 51.35477 5.076291 76.78919 7.739237 95.46322 11.52165 AD-519890.1 71.77843 5.504303 64.796 11.40644 87.89867 15.0868 91.90634 3.389965 AD-519929.1 51.24411 4.482367 39.80655 22.3568 46.70825 12.81069 67.24068 6.3593 AD-519931.1 50.30644 3.56686 35.76478 6.184685 39.3523 9.958837 59.53076 3.383324 AD-519932.1 31.44614 11.99819 32.37951 19.35007 37.62279 5.878389 55.21232 5.642967 AD-519933.1 32.84923 3.175817 32.48347 7.87957 71.16852 27.0411 78.68581 4.365061 AD-519935.2 23.19916 5.459604 31.95322 8.550425 40.71946 8.767387 70.20499 7.920042 AD-519942.1 33.43069 7.462724 50.81118 9.662815 71.10903 10.55588 83.13999 6.804144 AD-520005.1 54.87907 8.873501 69.27955 6.115951 85.07006 6.620292 88.0046 3.067445 AD-520006.1 24.04719 2.376527 98.12066 13.8079 81.4189 5.359304 87.97026 6.179845 AD-520007.1 16.0072 2.896706 65.2408 12.45506 76.75044 17.42631 89.26613 7.321347 AD-520008.1 70.54243 7.357645 90.52422 5.629111 112.8555 12.18067 110.4207 28.5502 AD-520011.1 61.34677 18.83765 96.91868 10.10156 98.16977 15.5554 112.8618 30.05178 AD-520020.1 56.73076 8.466195 47.01322 4.272449 58.86832 6.769924 90.48843 16.95068 AD-520021.1 58.05942 7.510224 25.28068 3.221174 43.14391 3.700495 77.34291 4.43031 AD-520022.1 60.33364 4.301973 72.83127 2.803794 84.01748 6.444848 91.72618 5.273958 AD-520023.1 53.1275 5.840407 73.45791 2.587847 93.04343 2.063577 109.9385 8.268382 AD-520024.1 37.0917 8.452936 64.47736 6.189783 71.63681 2.311097 101.2265 11.3285 AD-520025.1 36.99918 5.011139 101.3805 10.46419 99.36336 1.178845 125.1059 22.39854 AD-520034.1 50.74447 16.90927 101.1502 6.872769 102.561 8.055655 112.4464 30.31735 AD-520035.3 47.68497 13.2744 93.78555 3.138244 100.1176 8.06161 98.20826 14.06374 AD-520036.1 62.46816 8.270828 99.50618 4.35277 107.4029 6.917039 89.79064 10.87267 AD-520037.1 56.5259 8.816028 82.54556 13.37586 109.9246 4.245039 96.29902 5.863261 AD-520038.1 51.75385 18.57013 102.8392 8.317021 95.4829 1.332827 93.45705 10.27244 AD-520053.1 41.77559 8.867003 23.59732 7.625737 27.52882 4.75974 59.16995 5.396248 AD-520060.3 44.68219 8.424713 16.8034 1.660673 28.01705 2.276751 51.331 2.939989 AD-520061.3 28.67808 4.381034 13.29874 1.854016 37.21291 13.30954 52.88488 7.318163 AD-520064.5 48.04944 8.872461 16.34754 4.443817 26.32244 4.7663 59.63678 12.49412 AD-520065.4 66.23067 11.50931 16.51615 1.555023 22.67985 3.273242 50.31799 14.32623 AD-520066.2 95.65312 47.68023 18.82186 1.942078 24.67161 0.48992 39.66911 3.815141 AD-520067.3 105.1375 37.57514 16.24075 2.266037 21.7748 1.517695 47.79545 2.382244 AD-75289.4 56.22618 10.19969 24.54127 12.60091 39.53389 3.948394 64.83497 3.070942 AD-520068.3 70.91495 2.704445 27.78887 2.168692 40.12321 8.960175 74.079 7.777185 AD-520069.3 39.78116 11.92992 26.46514 3.712465 36.37967 5.03883 69.82406 8.52398 AD-520087.1 68.98191 8.459896 50.20663 11.44661 81.35279 14.63565 94.63964 15.23652 AD-520095.1 81.69757 9.807112 32.2698 2.79685 40.69272 3.69408 69.19573 1.070516 AD-520096.1 64.90297 1.936481 25.00834 2.580411 37.05238 5.070033 58.76053 3.332699 AD-520098.2 112.056 49.26435 28.88694 6.464563 34.33808 2.157562 62.1349 10.38576 AD-75256.2 75.79616 8.737346 47.65446 11.90689 64.17225 3.644674 87.85404 8.070264 AD-520099.3 59.03481 13.43313 25.29062 8.515026 37.29785 1.900832 72.75344 8.630579 AD-67575.8 35.92082 6.119518 32.1524 7.330741 32.35538 4.756249 69.60965 8.477324 AD-520101.3 58.99073 10.67199 28.93054 6.61911 36.35128 10.79135 84.78044 31.16482 AD-67605.8 59.32836 23.51253 23.70456 2.00817 37.35276 3.899334 64.87945 10.10013 AD-520102.1 75.87013 7.619236 41.39641 13.01122 61.67463 5.659235 87.90872 6.466547 AD-520103.1 82.82953 7.726372 80.61462 16.04746 90.10485 4.645091 84.60304 6.032456 AD-520123.1 102.8972 19.3969 23.28958 3.608176 33.1825 0.757984 58.49549 5.609074 AD-520127.1 73.10119 23.1491 34.41508 3.002116 66.96245 3.835703 89.71 5.774929 AD-520133.1 36.1541 6.391883 27.18259 3.059568 33.92563 6.050576 62.97858 8.296929 AD-520350.1 58.87999 14.59136 100.8357 1.826047 102.0606 4.816809 103.3828 15.06595 AD-520351.1 71.26303 7.088025 110.2778 18.72946 96.78415 8.786053 98.90987 17.25027 AD-520352.2 85.42707 13.84644 90.15955 6.926617 99.08475 1.971048 84.18354 2.660402 AD-520354.1 95.72204 19.82589 98.02466 11.36069 101.4274 6.452407 97.11452 14.81708 AD-520391.1 129.611 19.84147 99.27503 7.12102 100.53 8.937246 89.18045 4.171858 AD-520393.1 102.3696 22.0114 109.5158 13.74209 100.5261 11.22596 95.78871 5.496628 AD-520467.2 57.00049 5.905218 31.91459 2.380219 56.31104 4.401978 78.98328 3.377318 AD-520481.1 69.55206 1.607017 93.48413 3.90882 100.765 2.313415 105.4742 15.47609

Example 5. Structure-Activity Relationship Analyses

Based on the in vitro analyses in Example 4, structure-active relationship (SAR) analyses were performed. In particular, additional duplexes were designed, synthesized, and assayed in vitro and in vivo.

siRNAs were designed, synthesized, and prepared using methods known in the art and described above. In vitro screening assays in Hep3B and PCH cells with these siRNAs were performed as described above.

Detailed lists of the unmodified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 36. Detailed lists of the modified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 37.

The results of the transfection assays of the dsRNA agents listed in Tables 36 and 37 in Hep3B cells are shown in Table 38. The results of the transfection assays of the dsRNA agents listed in Tables 36 and 37 in primary cynomolgus hepatocytes (PCH) are shown in Table 39.

TABLE 36 Unmodified Sense and Antisense Strand Sequences of PNPLA3 dsRNA Agents SEQ ID Range in SEQ ID Range in Duplex Name Sense Sequence 5′ to 3′ NO: NM_025225.2 Antisense Sequence 5′ to 3′ NO: NM_025225.2 AD-519341.2 GCUACCCAUUAGGAUAAUGUU 2474 1208-1228 AACAUUAUCCUAAUGGGUAGCAA 2558 1206-1228 AD-519342.2 CUACCCAUUAGGAUAAUGUCU 2475 1209-1229 AGACAUUAUCCUAAUGGGUAGCA 2559 1207-1229 AD-519343.2 UACCCAUUAGGAUAAUGUCUU 2476 1210-1230 AAGACAUUAUCCUAAUGGGUAGC 2560 1208-1230 AD-1010713.1 ACCCAUUAGGAUAAUGUCUUA 2477 1145-1165 UAAGACAUUAUCCUAAUGGGUAG 2561 1143-1165 AD-519345.1 CCCAUUAGGAUAAUGUCUUAU 2478 1212-1232 AUAAGACAUUAUCCUAAUGGGUA 2562 1210-1232 AD-519346.1 CCAUUAGGAUAAUGUCUUAUU 2479 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 2563 1211-1233 AD-519347.1 CAUUAGGAUAAUGUCUUAUGU 2480 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 2564 1212-1234 AD-1010714.1 AUUAGGAUAAUGUCUUAUGUA 2481 1149-1169 UACAUAAGACAUUAUCCUAAUGG 2565 1147-1169 AD-1010715.1 UUAGGAUAAUGUCUUAUGUAA 2482 1150-1170 UUACAUAAGACAUUAUCCUAAUG 2566 1148-1170 AD-519350.2 UAGGAUAAUGUCUUAUGUAAU 2483 1217-1237 AUUACAUAAGACAUUAUCCUAAU 2567 1215-1237 AD-519351.7 AGGAUAAUGUCUUAUGUAAUU 2484 1218-1238 AAUUACAUAAGACAUUAUCCUAA 2568 1216-1238 AD-519352.1 GGAUAAUGUCUUAUGUAAUGU 2485 1219-1239 ACAUUACAUAAGACAUUAUCCUA 2569 1217-1239 AD-519353.1 GAUAAUGUCUUAUGUAAUGCU 2486 1220-1240 AGCAUUACAUAAGACAUUAUCCU 2570 1218-1240 AD-519354.1 AUAAUGUCUUAUGUAAUGCUU 2487 1221-1241 AAGCAUUACAUAAGACAUUAUCC 2571 1219-1241 AD-519355.1 UAAUGUCUUAUGUAAUGCUGU 2488 1222-1242 ACAGCAUUACAUAAGACAUUAUC 2572 1220-1242 AD-519356.1 AAUGUCUUAUGUAAUGCUGCU 2489 1223-1243 AGCAGCAUUACAUAAGACAUUAU 2573 1221-1243 AD-519357.1 AUGUCUUAUGUAAUGCUGCCU 2490 1224-1244 AGGCAGCAUUACAUAAGACAUUA 2574 1222-1244 AD-519358.1 UGUCUUAUGUAAUGCUGCCCU 2491 1225-1245 AGGGCAGCAUUACAUAAGACAUU 2575 1223-1245 AD-519359.1 GUCUUAUGUAAUGCUGCCCUU 2492 1226-1246 AAGGGCAGCAUUACAUAAGACAU 2576 1224-1246 AD-519360.1 UCUUAUGUAAUGCUGCCCUGU 2493 1227-1247 ACAGGGCAGCAUUACAUAAGACA 2577 1225-1247 AD-1010716.1 CUUAUGUAAUGCUGCCCUGUA 2494 1162-1182 UACAGGGCAGCAUUACAUAAGAC 2578 1160-1182 AD-519745.1 CCCAGCCUCUGAGCUGAGUUU 2495 1733-1753 AAACUCAGCUCAGAGGCUGGGAU 2579 1731-1753 AD-519746.1 CCAGCCUCUGAGCUGAGUUGU 2496 1734-1754 ACAACUCAGCUCAGAGGCUGGGA 2580 1732-1754 AD-519747.1 CAGCCUCUGAGCUGAGUUGGU 2497 1735-1755 ACCAACUCAGCUCAGAGGCUGGG 2581 1733-1755 AD-519748.1 AGCCUCUGAGCUGAGUUGGUU 2498 1736-1756 AACCAACUCAGCUCAGAGGCUGG 2582 1734-1756 AD-519749.1 GCCUCUGAGCUGAGUUGGUUU 2499 1737-1757 AAACCAACUCAGCUCAGAGGCUG 2583 1735-1757 AD-519750.1 CCUCUGAGCUGAGUUGGUUUU 2500 1738-1758 AAAACCAACUCAGCUCAGAGGCU 2584 1736-1758 AD-1010717.1 CUCUGAGCUGAGUUGGUUUUA 2501 1673-1693 UAAAACCAACUCAGCUCAGAGGC 2585 1671-1693 AD-67554.7 UCUGAGCUGAGUUGGUUUUAU 2502 1740-1760 AUAAAACCAACUCAGCUCAGAGG 2586 1738-1760 AD-519752.3 CUGAGCUGAGUUGGUUUUAUU 2503 1741-1761 AAUAAAACCAACUCAGCUCAGAG 2587 1739-1761 AD-1010718.1 UGAGCUGAGUUGGUUUUAUGA 2504 1676-1696 UCAUAAAACCAACUCAGCUCAGA 2588 1674-1696 AD-519754.9 GAGCUGAGUUGGUUUUAUGAU 2505 1743-1763 AUCAUAAAACCAACUCAGCUCAG 2589 1741-1763 AD-75276.2 AGCUGAGUUGGUUUUAUGAAA 2506 1744-1764 UUUCAUAAAACCAACUCAGCUCA 2590 1742-1764 AD-1010719.1 GCUGAGUUGGUUUUAUGAAAA 2507 1679-1699 UUUUCAUAAAACCAACUCAGCUC 2591 1677-1699 AD-519757.3 CUGAGUUGGUUUUAUGAAAAU 2508 1746-1766 AUUUUCAUAAAACCAACUCAGCU 2592 1744-1766 AD-519758.2 UGAGUUGGUUUUAUGAAAAGU 2509 1747-1767 ACUUUUCAUAAAACCAACUCAGC 2593 1745-1767 AD-519759.3 GAGUUGGUUUUAUGAAAAGCU 2510 1748-1768 AGCUUUUCAUAAAACCAACUCAG 2594 1746-1768 AD-67549.2 AGUUGGUUUUAUGAAAAGCUA 2511 1749-1769 UAGCUUUUCAUAAAACCAACUCA 2595 1747-1769 AD-519761.3 GUUGGUUUUAUGAAAAGCUAU 2512 1750-1770 AUAGCUUUUCAUAAAACCAACUC 2596 1748-1770 AD-519762.2 UUGGUUUUAUGAAAAGCUAGU 2513 1751-1771 ACUAGCUUUUCAUAAAACCAACU 2597 1749-1771 AD-1010720.1 UGGUUUUAUGAAAAGCUAGGA 2514 1686-1706 UCCUAGCUUUUCAUAAAACCAAC 2598 1684-1706 AD-67558.2 GGUUUUAUGAAAAGCUAGGAA 2515 1753-1773 UUCCUAGCUUUUCAUAAAACCAA 2599 1751-1773 AD-1010721.1 CUGCAAAGAUGAUAACCUUGA 2516 2034-2054 UCAAGGUUAUCAUCUUUGCAGAC 2600 2032-2054 AD-520009.1 UGCAAAGAUGAUAACCUUGAU 2517 2101-2121 AUCAAGGUUAUCAUCUUUGCAGA 2601 2099-2121 AD-520010.1 GCAAAGAUGAUAACCUUGACU 2518 2102-2122 AGUCAAGGUUAUCAUCUUUGCAG 2602 2100-2122 AD-1010722.1 CAAAGAUGAUAACCUUGACUA 2519 2037-2057 UAGUCAAGGUUAUCAUCUUUGCA 2603 2035-2057 AD-520012.1 AAAGAUGAUAACCUUGACUAU 2520 2104-2124 AUAGUCAAGGUUAUCAUCUUUGC 2604 2102-2124 AD-520013.1 AAGAUGAUAACCUUGACUACU 2521 2105-2125 AGUAGUCAAGGUUAUCAUCUUUG 2605 2103-2125 AD-1010723.1 AGAUGAUAACCUUGACUACUA 2522 2040-2060 UAGUAGUCAAGGUUAUCAUCUUU 2606 2038-2060 AD-1010724.1 GAUGAUAACCUUGACUACUAA 2523 2041-2061 UUAGUAGUCAAGGUUAUCAUCUU 2607 2039-2061 AD-1010725.1 AUGAUAACCUUGACUACUAAA 2524 2042-2062 UUUAGUAGUCAAGGUUAUCAUCU 2608 2040-2062 AD-1010726.1 UGAUAACCUUGACUACUAAAA 2525 2043-2063 UUUUAGUAGUCAAGGUUAUCAUC 2609 2041-2063 AD-520018.7 GAUAACCUUGACUACUAAAAU 2526 2110-2130 AUUUUAGUAGUCAAGGUUAUCAU 2610 2108-2130 AD-520019.1 AUAACCUUGACUACUAAAAAU 2527 2111-2131 AUUUUUAGUAGUCAAGGUUAUCA 2611 2109-2131 AD-520020.2 UAACCUUGACUACUAAAAACU 2528 2112-2132 AGUUUUUAGUAGUCAAGGUUAUC 2612 2110-2132 AD-520021.2 AACCUUGACUACUAAAAACGU 2529 2113-2133 ACGUUUUUAGUAGUCAAGGUUAU 2613 2111-2133 AD-520022.2 ACCUUGACUACUAAAAACGUU 2530 2114-2134 AACGUUUUUAGUAGUCAAGGUUA 2614 2112-2134 AD-520023.2 CCUUGACUACUAAAAACGUCU 2531 2115-2135 AGACGUUUUUAGUAGUCAAGGUU 2615 2113-2135 AD-520024.2 CUUGACUACUAAAAACGUCUU 2532 2116-2136 AAGACGUUUUUAGUAGUCAAGGU 2616 2114-2136 AD-520025.2 UUGACUACUAAAAACGUCUCU 2533 2117-2137 AGAGACGUUUUUAGUAGUCAAGG 2617 2115-2137 AD-1010727.1 UGACUACUAAAAACGUCUCCA 2534 2052-2072 UGGAGACGUUUUUAGUAGUCAAG 2618 2050-2072 AD-520027.1 GACUACUAAAAACGUCUCCAU 2535 2119-2139 AUGGAGACGUUUUUAGUAGUCAA 2619 2117-2139 AD-520028.1 ACUACUAAAAACGUCUCCAUU 2536 2120-2140 AAUGGAGACGUUUUUAGUAGUCA 2620 2118-2140 AD-520052.1 AUUUUAGAACACCUUUUUCAU 2537 2170-2190 AUGAAAAAGGUGUUCUAAAAUUA 2621 2168-2190 AD-520053.2 UUUUAGAACACCUUUUUCACU 2538 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 2622 2169-2191 AD-520054.1 UUUAGAACACCUUUUUCACCU 2539 2172-2192 AGGUGAAAAAGGUGUUCUAAAAU 2623 2170-2192 AD-1010728.1 UUAGAACACCUUUUUCACCUA 2540 2107-2127 UAGGUGAAAAAGGUGUUCUAAAA 2624 2105-2127 AD-1010729.1 UAGAACACCUUUUUCACCUAA 2541 2108-2128 UUAGGUGAAAAAGGUGUUCUAAA 2625 2106-2128 AD-520057.1 AGAACACCUUUUUCACCUAAU 2542 2175-2195 AUUAGGUGAAAAAGGUGUUCUAA 2626 2173-2195 AD-520058.1 GAACACCUUUUUCACCUAACU 2543 2176-2196 AGUUAGGUGAAAAAGGUGUUCUA 2627 2174-2196 AD-1010730.1 AACACCUUUUUCACCUAACUA 2544 2111-2131 UAGUUAGGUGAAAAAGGUGUUCU 2628 2109-2131 AD-1010731.1 ACACCUUUUUCACCUAACUAA 2545 2112-2132 UUAGUUAGGUGAAAAAGGUGUUC 2629 2110-2132 AD-1010732.1 CACCUUUUUCACCUAACUAAA 2546 2113-2133 UUUAGUUAGGUGAAAAAGGUGUU 2630 2111-2133 AD-520062.8 ACCUUUUUCACCUAACUAAAU 2547 2180-2200 AUUUAGUUAGGUGAAAAAGGUGU 2631 2178-2200 AD-520063.4 CCUUUUUCACCUAACUAAAAU 2548 2181-2201 AUUUUAGUUAGGUGAAAAAGGUG 2632 2179-2201 AD-1010733.1 CUUUUUCACCUAACUAAAAUA 2549 2116-2136 UAUUUUAGUUAGGUGAAAAAGGU 2633 2114-2136 AD-1010734.1 UUUUUCACCUAACUAAAAUAA 2550 2117-2137 UUAUUUUAGUUAGGUGAAAAAGG 2634 2115-2137 AD-520066.3 UUUUCACCUAACUAAAAUAAU 2551 2184-2204 AUUAUUUUAGUUAGGUGAAAAAG 2635 2182-2204 AD-520067.4 UUUCACCUAACUAAAAUAAUU 2552 2185-2205 AAUUAUUUUAGUUAGGUGAAAAA 2636 2183-2205 AD-75289.5 UUCACCUAACUAAAAUAAUGU 2553 2186-2206 ACAUUAUUUUAGUUAGGUGAAAA 2637 2184-2206 AD-520068.4 UCACCUAACUAAAAUAAUGUU 2554 2187-2207 AACAUUAUUUUAGUUAGGUGAAA 2638 2185-2207 AD-520069.4 CACCUAACUAAAAUAAUGUUU 2555 2188-2208 AAACAUUAUUUUAGUUAGGUGAA 2639 2186-2208 AD-1010735.1 ACCUAACUAAAAUAAUGUUUA 2556 2123-2143 UAAACAUUAUUUUAGUUAGGUGA 2640 2121-2143 AD-67584.6 CCUAACUAAAAUAAUGUUUAA 2557 2190-2210 UUAAACAUUAUUUUAGUUAGGUG 2641 2188-2210

TABLE 37 Modified Sense and Antisense Strand Sequencesof PNPLA3 dsRNA Agents Duplex Name Sense Sequence 5′ to 3′ SEQ ID NO: Antisense Sequence 5′ to 3′ SEQ ID NO: mRNATarget Sequence SEQ ID NO: AD-519341.2 gscsuaccCfaUfUfAfggauaauguuL96 2642 asAfscauUfaUfCfcuaaUfgGfguagcsasa 2726 UUGCUACCCAUUAGGAUAAUGUC 2810 AD-519342.2 csusacccAfuUfAfGfgauaaugucuL96 2643 asGfsacaUfuAfUfccuaAfuGfgguagscsa 2727 UGCUACCCAUUAGGAUAAUGUCU 2811 AD-519343.2 usascccaUfuAfGfGfauaaugucuuL96 2644 asAfsgacAfuUfAfuccuAfaUfggguasgsc 2728 GCUACCCAUUAGGAUAAUGUCUU 2812 AD-1010713.1 ascsccauUfaGfGfAfuaaugucuuaL96 2645 usAfsagaCfaUfUfauccUfaAfugggusasg 2729 CUACCCAUUAGGAUAAUGUCUUA 2813 AD-519345.1 cscscauuAfgGfAfUfaaugucuuauL96 2646 asUfsaagAfcAfUfuaucCfuAfaugggsusa 2730 UACCCAUUAGGAUAAUGUCUUAU 2814 AD-519346.1 cscsauuaGfgAfUfAfaugucuuauuL96 2647 asAfsuaaGfaCfAfuuauCfcUfaauggsgsu 2731 ACCCAUUAGGAUAAUGUCUUAUG 2815 AD-519347.1 csasuuagGfaUfAfAfugucuuauguL96 2648 asCfsauaAfgAfCfauuaUfcCfuaaugsgsg 2732 CCCAUUAGGAUAAUGUCUUAUGU 2816 AD-1010714.1 asusuaggAfuAfAfUfgucuuauguaL96 2649 usAfscauAfaGfAfcauuAfuCfcuaausgsg 2733 CCAUUAGGAUAAUGUCUUAUGUA 2817 AD-1010715.1 ususaggaUfaAfUfGfucuuauguaaL96 2650 usUfsacaUfaAfGfacauUfaUfccuaasusg 2734 CAUUAGGAUAAUGUCUUAUGUAA 2818 AD-519350.2 usasggauAfaUfGfUfcuuauguaauL96 2651 asUfsuacAfuAfAfgacaUfuAfuccuasasu 2735 AUUAGGAUAAUGUCUUAUGUAAU 2819 AD-519351.7 asgsgauaAfuGfUfCfuuauguaauuL96 2652 asAfsuuaCfaUfAfagacAfuUfauccusasa 2736 UUAGGAUAAUGUCUUAUGUAAUG 2820 AD-519352.1 gsgsauaaUfgUfCfUfuauguaauguL96 2653 asCfsauuAfcAfUfaagaCfaUfuauccsusa 2737 UAGGAUAAUGUCUUAUGUAAUGC 2821 AD-519353.1 gsasuaauGfuCfUfUfauguaaugcuL96 2654 asGfscauUfaCfAfuaagAfcAfuuaucscsu 2738 AGGAUAAUGUCUUAUGUAAUGCU 2822 AD-519354.1 asusaaugUfcUfUfAfuguaaugcuuL96 2655 asAfsgcaUfuAfCfauaaGfaCfauuauscsc 2739 GGAUAAUGUCUUAUGUAAUGCUG 2823 AD-519355.1 usasauguCfuUfAfUfguaaugcuguL96 2656 asCfsagcAfuUfAfcauaAfgAfcauuasusc 2740 GAUAAUGUCUUAUGUAAUGCUGC 2824 AD-519356.1 asasugucUfuAfUfGfuaaugcugcuL96 2657 asGfscagCfaUfUfacauAfaGfacauusasu 2741 AUAAUGUCUUAUGUAAUGCUGCC 2825 AD-519357.1 asusgucuUfaUfGfUfaaugcugccuL96 2658 asGfsgcaGfcAfUfuacaUfaAfgacaususa 2742 UAAUGUCUUAUGUAAUGCUGCCC 2826 AD-519358.1 usgsucuuAfuGfUfAfaugcugcccuL96 2659 asGfsggcAfgCfAfuuacAfuAfagacasusu 2743 AAUGUCUUAUGUAAUGCUGCCCU 2827 AD-519359.1 gsuscuuaUfgUfAfAfugcugcccuuL96 2660 asAfsgggCfaGfCfauuaCfaUfaagacsasu 2744 AUGUCUUAUGUAAUGCUGCCCUG 2828 AD-519360.1 uscsuuauGfuAfAfUfgcugcccuguL96 2661 asCfsaggGfcAfGfcauuAfcAfuaagascsa 2745 UGUCUUAUGUAAUGCUGCCCUGU 2829 AD-1010716.1 csusuaugUfaAfUfGfcugcccuguaL96 2662 usAfscagGfgCfAfgcauUfaCfauaagsasc 2746 GUCUUAUGUAAUGCUGCCCUGUA 2830 AD-519745.1 cscscagcCfuCfUfGfagcugaguuuL96 2663 asAfsacuCfaGfCfucagAfgGfcugggsasu 2747 AUCCCAGCCUCUGAGCUGAGUUG 2831 AD-519746.1 cscsagccUfcUfGfAfgcugaguuguL96 2664 asCfsaacUfcAfGfcucaGfaGfgcuggsgsa 2748 UCCCAGCCUCUGAGCUGAGUUGG 2832 AD-519747.1 csasgccuCfuGfAfGfcugaguugguL96 2665 asCfscaaCfuCfAfgcucAfgAfggcugsgsg 2749 CCCAGCCUCUGAGCUGAGUUGGU 2833 AD-519748.1 asgsccucUfgAfGfCfugaguugguuL96 2666 asAfsccaAfcUfCfagcuCfaGfaggcusgsg 2750 CCAGCCUCUGAGCUGAGUUGGUU 2834 AD-519749.1 gscscucuGfaGfCfUfgaguugguuuL96 2667 asAfsaccAfaCfUfcagcUfcAfgaggcsusg 2751 CAGCCUCUGAGCUGAGUUGGUUU 2835 AD-519750.1 cscsucugAfgCfUfGfaguugguuuuL96 2668 asAfsaacCfaAfCfucagCfuCfagaggscsu 2752 AGCCUCUGAGCUGAGUUGGUUUU 2836 AD-1010717.1 csuscugaGfcUfGfAfguugguuuuaL96 2669 usAfsaaaCfcAfAfcucaGfcUfcagagsgsc 2753 GCCUCUGAGCUGAGUUGGUUUUA 2837 AD-67554.7 uscsugagCfuGfAfGfuugguuuuauL96 2670 asUfsaaaAfcCfAfacucAfgCfucagasgsg 2754 CCUCUGAGCUGAGUUGGUUUUAU 2838 AD-519752.3 csusgagcUfgAfGfUfugguuuuauuL96 2671 asAfsuaaAfaCfCfaacuCfaGfcucagsasg 2755 CUCUGAGCUGAGUUGGUUUUAUG 2839 AD-1010718.1 usgsagcuGfaGfUfUfgguuuuaugaL96 2672 usCfsauaAfaAfCfcaacUfcAfgcucasgsa 2756 UCUGAGCUGAGUUGGUUUUAUGA 2840 AD-519754.9 gsasgcugAfgUfUfGfguuuuaugauL96 2673 asUfscauAfaAfAfccaaCfuCfagcucsasg 2757 CUGAGCUGAGUUGGUUUUAUGAA 2841 AD-75276.2 asgscugaGfuUfGfGfuuuuaugaaaL96 2674 usUfsucaUfaAfAfaccaAfcUfcagcuscsa 2758 UGAGCUGAGUUGGUUUUAUGAAA 2842 AD-1010719.1 gscsugagUfuGfGfUfuuuaugaaaaL96 2675 usUfsuucAfuAfAfaaccAfaCfucagcsusc 2759 GAGCUGAGUUGGUUUUAUGAAAA 2843 AD-519757.3 csusgaguUfgGfUfUfuuaugaaaauL96 2676 asUfsuuuCfaUfAfaaacCfaAfcucagscsu 2760 AGCUGAGUUGGUUUUAUGAAAAG 2844 AD-519758.2 usgsaguuGfgUfUfUfuaugaaaaguL96 2677 asCfsuuuUfcAfUfaaaaCfcAfacucasgsc 2761 GCUGAGUUGGUUUUAUGAAAAGC 2845 AD-519759.3 gsasguugGfuUfUfUfaugaaaagcuL96 2678 asGfscuuUfuCfAfuaaaAfcCfaacucsasg 2762 CUGAGUUGGUUUUAUGAAAAGCU 2846 AD-67549.2 asgsuuggUfuUfUfAfugaaaagcuaL96 2679 usAfsgcuUfuUfCfauaaAfaCfcaacuscsa 2763 UGAGUUGGUUUUAUGAAAAGCUA 2847 AD-519761.3 gsusugguUfuUfAfUfgaaaagcuauL96 2680 asUfsagcUfuUfUfcauaAfaAfccaacsusc 2764 GAGUUGGUUUUAUGAAAAGCUAG 2848 AD-519762.2 ususgguuUfuAfUfGfaaaagcuaguL96 2681 asCfsuagCfuUfUfucauAfaAfaccaascsu 2765 AGUUGGUUUUAUGAAAAGCUAGG 2849 AD-1010720.1 usgsguuuUfaUfGfAfaaagcuaggaL96 2682 usCfscuaGfcUfUfuucaUfaAfaaccasasc 2766 GUUGGUUUUAUGAAAAGCUAGGA 2850 AD-67558.2 gsgsuuuuAfuGfAfAfaagcuaggaaL96 2683 usUfsccuAfgCfUfuuucAfuAfaaaccsasa 2767 UUGGUUUUAUGAAAAGCUAGGAA 2851 AD-1010721.1 csusgcaaAfgAfUfGfauaaccuugaL96 2684 usCfsaagGfuUfAfucauCfuUfugcagsasc 2768 GUCUGCAAAGAUGAUAACCUUGA 2852 AD-520009.1 usgscaaaGfaUfGfAfuaaccuugauL96 2685 asUfscaaGfgUfUfaucaUfcUfuugcasgsa 2769 UCUGCAAAGAUGAUAACCUUGAC 2853 AD-520010.1 gscsaaagAfuGfAfUfaaccuugacuL96 2686 asGfsucaAfgGfUfuaucAfuCfuuugcsasg 2770 CUGCAAAGAUGAUAACCUUGACU 2854 AD-1010722.1 csasaagaUfgAfUfAfaccuugacuaL96 2687 usAfsgucAfaGfGfuuauCfaUfcuuugscsa 2771 UGCAAAGAUGAUAACCUUGACUA 2855 AD-520012.1 asasagauGfaUfAfAfccuugacuauL96 2688 asUfsaguCfaAfGfguuaUfcAfucuuusgsc 2772 GCAAAGAUGAUAACCUUGACUAC 2856 AD-520013.1 asasgaugAfuAfAfCfcuugacuacuL96 2689 asGfsuagUfcAfAfgguuAfuCfaucuususg 2773 CAAAGAUGAUAACCUUGACUACU 2857 AD-1010723.1 asgsaugaUfaAfCfCfuugacuacuaL96 2690 usAfsguaGfuCfAfagguUfaUfcaucususu 2774 AAAGAUGAUAACCUUGACUACUA 2858 AD-1010724.1 gsasugauAfaCfCfUfugacuacuaaL96 2691 usUfsaguAfgUfCfaaggUfuAfucaucsusu 2775 AAGAUGAUAACCUUGACUACUAA 2859 AD-1010725.1 asusgauaAfcCfUfUfgacuacuaaaL96 2692 usUfsuagUfaGfUfcaagGfuUfaucauscsu 2776 AGAUGAUAACCUUGACUACUAAA 2860 AD-1010726.1 usgsauaaCfcUfUfGfacuacuaaaaL96 2693 usUfsuuaGfuAfGfucaaGfgUfuaucasusc 2777 GAUGAUAACCUUGACUACUAAAA 2861 AD-520018.7 gsasuaacCfuUfGfAfcuacuaaaauL96 2694 asUfsuuuAfgUfAfgucaAfgGfuuaucsasu 2778 AUGAUAACCUUGACUACUAAAAA 2862 AD-520019.1 asusaaccUfuGfAfCfuacuaaaaauL96 2695 asUfsuuuUfaGfUfagucAfaGfguuauscsa 2779 UGAUAACCUUGACUACUAAAAAC 2863 AD-520020.2 usasaccuUfgAfCfUfacuaaaaacuL96 2696 asGfsuuuUfuAfGfuaguCfaAfgguuasusc 2780 GAUAACCUUGACUACUAAAAACG 2864 AD-520021.2 asasccuuGfaCfUfAfcuaaaaacguL96 2697 asCfsguuUfuUfAfguagUfcAfagguusasu 2781 AUAACCUUGACUACUAAAAACGU 2865 AD-520022.2 ascscuugAfcUfAfCfuaaaaacguuL96 2698 asAfscguUfuUfUfaguaGfuCfaaggususa 2782 UAACCUUGACUACUAAAAACGUC 2866 AD-520023.2 cscsuugaCfuAfCfUfaaaaacgucuL96 2699 asGfsacgUfuUfUfuaguAfgUfcaaggsusu 2783 AACCUUGACUACUAAAAACGUCU 2867 AD-520024.2 csusugacUfaCfUfAfaaaacgucuuL96 2700 asAfsgacGfuUfUfuuagUfaGfucaagsgsu 2784 ACCUUGACUACUAAAAACGUCUC 2868 AD-520025.2 ususgacuAfcUfAfAfaaacgucucuL96 2701 asGfsagaCfgUfUfuuuaGfuAfgucaasgsg 2785 CCUUGACUACUAAAAACGUCUCC 2869 AD-1010727.1 usgsacuaCfuAfAfAfaacgucuccaL96 2702 usGfsgagAfcGfUfuuuuAfgUfagucasasg 2786 CUUGACUACUAAAAACGUCUCCA 2870 AD-520027.1 gsascuacUfaAfAfAfacgucuccauL96 2703 asUfsggaGfaCfGfuuuuUfaGfuagucsasa 2787 UUGACUACUAAAAACGUCUCCAU 2871 AD-520028.1 ascsuacuAfaAfAfAfcgucuccauuL96 2704 asAfsuggAfgAfCfguuuUfuAfguaguscsa 2788 UGACUACUAAAAACGUCUCCAUG 2872 AD-520052.1 asusuuuaGfaAfCfAfccuuuuucauL96 2705 asUfsgaaAfaAfGfguguUfcUfaaaaususa 2789 UAAUUUUAGAACACCUUUUUCAC 2873 AD-520053.2 ususuuagAfaCfAfCfcuuuuucacuL96 2706 asGfsugaAfaAfAfggugUfuCfuaaaasusu 2790 AAUUUUAGAACACCUUUUUCACC 2874 AD-520054.1 ususuagaAfcAfCfCfuuuuucaccuL96 2707 asGfsgugAfaAfAfagguGfuUfcuaaasasu 2791 AUUUUAGAACACCUUUUUCACCU 2875 AD-1010728.1 ususagaaCfaCfCfUfuuuucaccuaL96 2708 usAfsgguGfaAfAfaaggUfgUfucuaasasa 2792 UUUUAGAACACCUUUUUCACCUA 2876 AD-1010729.1 usasgaacAfcCfUfUfuuucaccuaaL96 2709 usUfsaggUfgAfAfaaagGfuGfuucuasasa 2793 UUUAGAACACCUUUUUCACCUAA 2877 AD-520057.1 asgsaacaCfcUfUfUfuucaccuaauL96 2710 asUfsuagGfuGfAfaaaaGfgUfguucusasa 2794 UUAGAACACCUUUUUCACCUAAC 2878 AD-520058.1 gsasacacCfuUfUfUfucaccuaacuL96 2711 asGfsuuaGfgUfGfaaaaAfgGfuguucsusa 2795 UAGAACACCUUUUUCACCUAACU 2879 AD-1010730.1 asascaccUfuUfUfUfcaccuaacuaL96 2712 usAfsguuAfgGfUfgaaaAfaGfguguuscsu 2796 AGAACACCUUUUUCACCUAACUA 2880 AD-1010731.1 ascsaccuUfuUfUfCfaccuaacuaaL96 2713 usUfsaguUfaGfGfugaaAfaAfggugususc 2797 GAACACCUUUUUCACCUAACUAA 2881 AD-1010732.1 csasccuuUfuUfCfAfccuaacuaaaL96 2714 usUfsuagUfuAfGfgugaAfaAfaggugsusu 2798 AACACCUUUUUCACCUAACUAAA 2882 AD-520062.8 ascscuuuUfuCfAfCfcuaacuaaauL96 2715 asUfsuuaGfuUfAfggugAfaAfaaggusgsu 2799 ACACCUUUUUCACCUAACUAAAA 2883 AD-520063.4 cscsuuuuUfcAfCfCfuaacuaaaauL96 2716 asUfsuuuAfgUfUfagguGfaAfaaaggsusg 2800 CACCUUUUUCACCUAACUAAAAU 2884 AD-1010733.1 csusuuuuCfaCfCfUfaacuaaaauaL96 2717 usAfsuuuUfaGfUfuaggUfgAfaaaagsgsu 2801 ACCUUUUUCACCUAACUAAAAUA 2885 AD-1010734.1 ususuuucAfcCfUfAfacuaaaauaaL96 2718 usUfsauuUfuAfGfuuagGfuGfaaaaasgsg 2802 CCUUUUUCACCUAACUAAAAUAA 2886 AD-520066.3 ususuucaCfcUfAfAfcuaaaauaauL96 2719 asUfsuauUfuUfAfguuaGfgUfgaaaasasg 2803 CUUUUUCACCUAACUAAAAUAAU 2887 AD-520067.4 ususucacCfuAfAfCfuaaaauaauuL96 2720 asAfsuuaUfuUfUfaguuAfgGfugaaasasa 2804 UUUUUCACCUAACUAAAAUAAUG 2888 AD-75289.5 ususcaccUfaAfCfUfaaaauaauguL96 2721 asCfsauuAfuUfUfuaguUfaGfgugaasasa 2805 UUUUCACCUAACUAAAAUAAUGU 2889 AD-520068.4 uscsaccuAfaCfUfAfaaauaauguuL96 2722 asAfscauUfaUfUfuuagUfuAfggugasasa 2806 UUUCACCUAACUAAAAUAAUGUU 2890 AD-520069.4 csasccuaAfcUfAfAfaauaauguuuL96 2723 asAfsacaUfuAfUfuuuaGfuUfaggugsasa 2807 UUCACCUAACUAAAAUAAUGUUU 2891 AD-1010735.1 ascscuaaCfuAfAfAfauaauguuuaL96 2724 usAfsaacAfuUfAfuuuuAfgUfuaggusgsa 2808 UCACCUAACUAAAAUAAUGUUUA 2892 AD-67584.6 cscsuaacUfaAfAfAfuaauguuuaaL96 2725 usUfsaaaCfaUfUfauuuUfaGfuuaggsusg 2809 CACCUAACUAAAAUAAUGUUUAA 2893

TABLE 38 PNPLA3 Single Dose Screen in Hep3B Cells 50 nM 10 nM 1 nM 0.1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-519341.2 52.21351 9.72399 66.63808 2.895261 115.7241 21.02387 107.1347 9.759609 AD-519342.2 37.71079 3.794796 65.62938 11.05422 102.1424 16.39283 103.5589 10.61437 AD-519343.2 25.96845 4.710585 37.97371 3.607549 107.4286 25.18447 113.9497 11.86309 AD-1010713.1 34.60493 14.82067 36.59308 5.337726 70.32418 8.024292 78.80994 4.447829 AD-519345.1 22.32288 9.868964 25.18423 5.868495 65.10216 2.459937 94.87672 13.77126 AD-519346.1 20.26202 4.882502 24.92529 9.086903 59.48022 11.21961 88.28443 6.544299 AD-519347.1 19.10368 3.401874 24.4461 3.123502 48.07209 18.58102 83.26504 3.650574 AD-1010714.1 31.37001 9.990933 36.67566 2.271094 69.29489 15.01823 74.98269 12.36223 AD-1010715.1 16.19338 3.256379 25.12577 5.098789 60.85548 8.686738 85.44148 14.72008 AD-519350.2 28.06864 12.63968 32.84701 4.418529 82.48389 15.13772 93.39421 13.52473 AD-519351.7 33.88484 16.17628 33.78251 6.620053 71.89585 15.80254 90.4867 7.497389 AD-519352.1 40.65545 6.19204 60.36501 10.17285 113.2654 7.292487 109.1059 15.1724 AD-519353.1 44.81905 17.20036 47.75754 1.320646 94.73342 8.394781 90.02693 9.494433 AD-519354.1 34.56728 6.889712 35.39008 5.779131 96.93029 26.32896 78.94062 15.65334 AD-519355.1 36.34872 3.664761 39.64831 11.73524 74.37666 10.15151 92.14636 20.76655 AD-519356.1 39.22631 11.96501 41.77619 4.720679 113.64 5.511916 86.95401 15.26991 AD-519357.1 59.98484 12.63175 77.68085 9.16923 147.7793 20.55612 122.0946 16.731 AD-519358.1 80.93125 19.23246 104.5258 17.88605 168.0363 46.44965 125.4269 11.7382 AD-519359.1 47.93771 19.70146 54.48883 6.769038 120.8681 28.98598 113.0698 18.87589 AD-519360.1 51.88021 2.305568 79.62266 11.70936 127.4202 14.54277 104.4351 16.67458 AD-1010716.1 61.44569 7.470191 75.03329 9.85015 120.3619 11.72527 92.32831 8.962171 AD-519745.1 54.08626 4.474803 66.53918 7.473265 93.45095 19.42486 89.00871 18.53944 AD-519746.1 61.75012 16.4376 68.23892 8.131801 117.0866 22.06383 83.27749 11.83074 AD-519747.1 54.70269 7.405541 78.41627 22.95503 159.082 33.22542 127.5855 7.255807 AD-519748.1 65.97231 12.64665 75.88878 13.07825 132.7913 7.238703 121.5269 6.610075 AD-519749.1 53.18769 11.52972 60.79999 7.719713 104.5054 17.13132 109.7383 20.90164 AD-519750.1 43.39469 4.611303 49.77677 7.234957 95.78512 18.21308 89.9017 12.82374 AD-1010717.1 40.17121 3.648584 49.80754 8.559616 103.4496 28.49196 88.25619 12.35317 AD-67554.7 29.95814 2.516048 41.39102 2.186988 80.61999 14.18474 80.36522 12.59649 AD-519752.3 35.50279 6.566427 44.05242 5.616278 70.0757 7.854597 67.1874 8.699813 AD-1010718.1 53.91995 6.177942 49.79666 7.576005 117.297 23.82981 132.2529 18.60175 AD-519754.9 63.50346 10.99599 81.53427 7.655464 137.8708 5.336041 150.6811 23.78481 AD-75276.2 43.76833 3.39359 54.27132 12.55485 110.3757 32.69547 93.1702 15.68035 AD-1010719.1 47.69971 11.07692 59.21313 4.434726 106.8359 19.33077 103.6515 11.85168 AD-519757.3 33.85014 2.34844 50.08412 5.579596 74.93986 9.466204 86.56796 5.483717 AD-519758.2 43.8025 3.987581 45.81134 7.638643 86.37357 3.983253 84.44134 7.159128 AD-519759.3 49.92008 6.958198 65.85982 22.90039 92.37066 20.62228 88.02301 11.24555 AD-67549.2 40.81684 7.036935 41.18702 2.409983 79.36188 24.53416 75.45676 3.921288 AD-519761.3 52.59379 9.420365 60.05315 6.050376 110.6665 15.70977 158.6849 22.7737 AD-519762.2 62.42972 6.590522 78.48425 9.982384 156.4083 30.45582 138.6577 14.49926 AD-1010720.1 64.18533 2.151443 73.60328 8.131667 142.6825 22.50819 122.0853 35.25396 AD-67558.2 59.75756 10.22261 73.57273 19.67153 138.1327 36.98034 104.997 8.008329 AD-1010721.1 74.90593 15.45991 82.13895 5.893665 109.8963 16.77037 101.386 9.636034 AD-520009.1 50.61456 5.090165 56.27916 5.404256 100.9592 34.02186 88.93466 11.18383 AD-520010.1 33.02608 4.744807 47.43298 8.950835 75.80949 13.90891 75.48906 4.702905 AD-1010722.1 63.11917 8.123617 52.23517 6.720795 125.0262 41.7076 125.9861 23.49933 AD-520012.1 30.9272 6.526604 43.07578 11.16706 88.39469 12.68997 103.7798 24.49013 AD-520013.1 41.9009 4.238882 63.29151 7.125221 124.7904 34.66747 104.344 13.70864 AD-1010723.1 43.01932 10.6476 72.63283 17.44516 114.8704 43.33915 105.7161 27.04454 AD-1010724.1 26.91853 1.618019 38.78679 2.813816 78.07498 17.52199 85.7066 11.74145 AD-1010725.1 36.41932 6.514834 47.58067 9.390825 71.49476 10.14546 74.93561 9.65005 AD-1010726.1 26.60712 4.09594 42.27913 12.7339 47.18645 17.81605 73.13987 7.894395 AD-520018.7 18.44357 6.262576 29.11779 3.214319 66.64326 36.12656 65.92984 5.919764 AD-520019.1 30.9879 6.706704 40.51287 1.315161 110.6345 37.86973 132.9561 24.06574 AD-520020.2 43.19306 6.866604 53.59392 12.8242 130.4697 25.45931 147.5214 38.32286 AD-520021.2 45.2373 5.708867 56.12506 5.681373 107.7844 21.34967 109.4796 4.555275 AD-520022.2 44.38727 3.592447 63.13264 9.558727 90.93577 6.741281 122.2097 7.654652 AD-520023.2 44.84941 4.670034 68.76309 3.408301 110.8257 11.2628 96.42404 6.713721 AD-520024.2 26.61322 1.991881 44.74325 7.635738 86.37162 27.17556 87.32308 11.92624 AD-520025.2 30.00974 5.75711 49.41303 8.124655 63.23468 31.2429 79.01933 12.73629 AD-1010727.1 40.32772 11.51333 50.63384 4.770684 84.37563 13.33681 87.94465 21.35558 AD-520027.1 50.58705 4.549675 63.91679 8.228882 134.9979 10.39521 116.9236 21.88092 AD-520028.1 48.1695 23.15682 56.18631 9.338642 118.8548 28.85886 136.3715 13.31997 AD-520052.1 35.48123 3.572863 52.33214 7.956613 101.3041 18.89961 117.0929 16.54513 AD-520053.2 25.61558 10.29700 38.30151 6.369982 68.81292 6.915711 82.94108 12.72222 AD-520054.1 53.65793 9.751686 71.48438 8.270167 113.3745 15.87967 98.42625 21.29711 AD-1010728.1 32.80095 10.45869 37.65466 2.605574 75.27318 8.757435 78.84613 9.210613 AD-1010729.1 22.0078 5.293667 32.18646 4.565428 68.33047 13.32244 67.99557 7.219904 AD-520057.1 38.52799 10.39505 38.28161 5.571138 75.44463 10.42506 103.8287 15.21887 AD-520058.1 59.14763 10.92649 59.8011 4.162056 114.1316 37.25307 141.0098 10.53144 AD-1010730.1 40.4781 6.024048 55.60748 3.680185 100.0228 27.00547 112.4548 23.44009 AD-1010731.1 24.90774 6.30736 37.25631 6.906268 77.26408 2.016841 80.71451 8.743286 AD-1010732.1 22.08367 3.888774 31.15355 4.133469 77.83024 21.58935 80.54815 2.883486 AD-520062.8 18.81467 2.512378 36.65627 4.469572 66.14384 9.074255 60.44039 8.883144 AD-520063.4 26.08143 3.912924 30.58296 2.004512 62.57235 12.42262 61.85164 3.906668 AD-1010733.1 28.41876 6.38097 36.49689 5.497472 69.50661 8.535372 97.67116 16.23765 AD-1010734.1 39.26943 2.250103 41.78536 2.940417 73.32514 8.491441 113.9301 16.01803 AD-520066.3 53.94827 7.168949 63.71949 12.08307 84.23585 9.556703 103.0603 12.6162 AD-520067.4 47.7984 18.05231 43.02687 12.78031 79.26626 20.89056 91.82778 14.74887 AD-75289.5 47.40988 6.156409 56.54605 7.093207 105.8225 20.87666 103.5825 8.552423 AD-520068.4 75.24691 7.353686 61.79409 10.56019 106.4331 12.04378 131.3598 100.0784 AD-520069.4 49.35648 17.62018 54.35528 7.380401 87.31947 8.168188 75.96545 3.57541 AD-1010735.1 24.390444 6.60858 29.219511 5.6575855 58.0864 16.974511 61.029022 7.29078 AD-67584.6 33.49671 5.09947 29.77183 1.370337 61.85995 3.782581 84.85692 13.49881

TABLE 39 PNPLA3 Single Dose Screen in Primary Cynomolgus Hepatocytes (PCH) 50 nM 10 nM 1 nM 0.1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-519341.2 33.18084 4.704386 51.77473 11.5752 102.7701 28.44004 86.5927 19.77298 AD-519342.2 18.58562 2.337486 36.98398 12.37523 72.89022 19.41867 88.26393 11.36751 AD-519343.2 14.92807 4.226682 25.44275 6.942827 34.02826 9.334955 78.65741 29.73611 AD-1010713.1 17.54145 2.864549 16.87213 0.624218 31.41544 9.277138 46.05722 5.815894 AD-519345.1 13.0926 1.627968 17.73351 4.187537 33.40954 11.52569 61.76661 12.4429 AD-519346.1 10.6233 1.207162 15.80567 3.743256 25.06314 8.677675 50.53195 18.40728 AD-519347.1 12.13834 1.973554 16.73735 3.727868 29.75023 6.687112 72.91558 28.98494 AD-1010714.1 22.58467 5.729209 42.90185 5.650413 53.69003 13.75494 106.2292 31.96262 AD-1010715.1 30.47287 2.033359 42.77617 12.49897 48.47358 7.69011 136.89 52.34067 AD-519350.2 21.67436 0.892886 30.51011 13.75843 33.43008 3.661736 64.39207 3.609213 AD-519351.7 38.4739 3.248472 45.75867 7.169571 42.10825 4.048311 73.02853 7.464011 AD-519352.1 22.72899 1.379361 20.50615 1.899178 41.65781 2.492385 81.90975 7.028732 AD-519353.1 31.25338 6.519443 40.64245 8.24087 56.83054 9.822431 89.05521 5.409826 AD-519354.1 23.21968 0.525641 32.99807 6.969361 35.03392 5.055374 66.52434 5.345017 AD-519355.1 23.4425 3.045451 29.1657 11.45662 34.05687 10.96159 68.50802 3.06385 AD-519356.1 38.10175 10.02499 45.20675 10.47852 72.97533 11.00292 93.51506 0.458343 AD-519357.1 68.10435 5.028706 73.60291 12.47881 113.0562 9.035648 123.3918 14.48132 AD-519358.1 109.7343 17.92171 120.1505 13.91801 101.5 15.15514 102.0346 7.956678 AD-519359.1 30.44511 3.278255 29.70762 5.861745 61.38724 5.037295 84.23748 4.7701 AD-519360.1 71.32011 6.26144 60.64032 5.051455 94.20443 20.83632 105.3515 17.96005 AD-1010716.1 41.7096 3.055802 41.9722 9.091862 65.15609 12.01425 95.19302 7.29294 AD-519745.1 43.96581 6.84819 41.29717 11.96036 57.69556 3.294909 101.7143 27.79148 AD-519746.1 47.49997 4.691397 49.42224 10.30268 69.45745 1.687739 106.0236 34.1365 AD-519747.1 53.52203 9.621605 51.35867 12.09421 88.54727 16.1145 104.6844 15.33743 AD-519748.1 35.72106 1.970757 44.39531 12.37111 78.95901 31.30559 93.55954 7.035535 AD-519749.1 30.27302 2.186866 35.64525 6.3804 46.6698 13.31953 86.65986 15.2604 AD-519750.1 25.55223 1.015786 38.29566 5.873705 42.79628 11.71471 83.33708 17.28221 AD-1010717.1 22.19581 0.444081 28.21198 14.87198 28.17989 2.0589 63.12416 12.68905 AD-67554.7 20.5347 2.086194 24.32709 7.309121 24.89249 2.097502 64.26467 26.77638 AD-519752.3 25.67992 1.931366 25.38746 5.112742 42.34479 11.2942 75.52802 10.90585 AD-1010718.1 27.18608 3.715747 44.69634 13.64543 48.87007 19.38281 78.41101 3.841614 AD-519754.9 24.58058 2.85202 28.55926 7.04754 47.1156 7.0551 71.75772 5.289018 AD-75276.2 19.94154 0.715945 24.59512 8.783805 30.73496 3.219686 51.97564 0.855416 AD-1010719.1 18.46757 1.784953 26.25269 12.98876 26.24527 1.8937 53.23716 8.284764 AD-519757.3 17.83558 1.13252 17.56734 2.632221 24.14466 6.652008 58.05775 6.720028 AD-519758.2 30.32832 14.98666 34.33039 8.904242 41.55164 3.189487 70.087 8.848411 AD-519759.3 27.61483 3.201485 28.96866 9.441592 51.66033 7.747181 84.45716 2.659745 AD-67549.2 25.26417 3.325598 24.0704 8.074096 57.06476 29.59065 66.76109 6.859419 AD-519761.3 27.29948 4.061702 29.40234 8.2302 55.97938 17.49415 73.8286 14.32816 AD-519762.2 26.2541 2.459677 29.08398 11.56778 47.0892 6.508957 71.65844 6.793264 AD-1010720.1 35.10105 11.0271 54.50536 17.15505 60.95521 6.424327 90.65354 1.622368 AD-67558.2 27.26775 1.703701 31.83117 3.376294 48.59497 11.6838 80.23711 9.139227 AD-1010721.1 98.73488 10.67008 88.63852 7.615453 93.6516 8.708677 115.0284 13.90528 AD-520009.1 115.1881 12.30103 89.73245 7.894185 118.2456 7.323628 95.74807 5.145745 AD-520010.1 79.89452 5.894902 73.57593 14.79716 77.45514 10.3914 94.76517 5.195967 AD-1010722.1 109.3595 4.505885 100.8274 14.68297 95.49468 15.87513 100.5743 0 AD-520012.1 97.07213 20.47878 91.76029 9.692549 81.80022 11.24291 94.5523 12.13118 AD-520013.1 103.4489 3.943599 108.8068 18.33588 109.6015 12.80929 93.2641 4.523871 AD-1010723.1 27.58843 2.338596 35.65611 10.1328 47.56411 9.976614 90.99518 25.74328 AD-1010724.1 26.46728 1.47565 26.90976 9.425383 41.29731 5.581072 82.42602 17.91712 AD-1010725.1 80.0121 1.320144 71.03697 8.193035 82.02665 7.278687 88.37711 12.41996 AD-1010726.1 63.49658 3.453642 62.83583 7.301534 61.38155 2.787238 79.46593 6.682405 AD-520018.7 19.27382 1.035111 23.71918 6.107674 42.88368 15.39814 51.91096 6.597352 AD-520019.1 67.75371 4.815317 85.46413 27.97837 71.69335 10.2165 76.65592 7.666444 AD-520020.2 49.64119 2.559106 51.89577 10.90042 64.34529 12.73844 78.40491 2.962168 AD-520021.2 32.19649 2.923462 38.97851 7.033731 44.36721 4.429857 83.14057 9.906269 AD-520022.2 88.52092 3.683218 80.57782 9.474541 85.40397 11.73205 85.33902 19.51987 AD-520023.2 80.54776 7.363073 79.91236 12.3369 85.55517 8.321525 92.01758 8.16751 AD-520024.2 70.48732 4.496364 68.08949 12.0069 63.8255 4.218374 86.79899 6.014172 AD-520025.2 106.695 7.559647 104.8586 8.904212 91.01226 5.278714 95.25508 5.172506 AD-1010727.1 84.29834 2.385382 79.63431 13.52425 79.97708 9.775323 107.7104 11.7926 AD-520027.1 105.0338 10.42553 128.6891 32.45594 91.12301 5.761488 88.55991 5.638937 AD-520028.1 84.80983 4.623143 91.43452 13.36049 74.04805 14.08942 86.91035 10.10266 AD-520052.1 59.00281 2.122686 70.00039 17.00761 69.18847 13.57631 85.22323 3.765275 AD-520053.2  15.3949 1.833054 21.17127 6.166623 26.30001 1.075899 49.58136 3.127215 AD-520054.1 30.39768 1.557474 28.43912 1.44968 62.62253 14.75011 80.48458 6.455693 AD-1010728.1 27.49196 18.51724 22.77339 8.901944 27.00153 3.445434 54.65515 5.160433 AD-1010729.1 27.52835 1.075856 36.00578 17.45302 46.0388 21.7126 69.47384 16.70382 AD-520057.1 27.20295 3.827477 39.78196 17.8445 61.48804 29.47533 44.24807 1.517811 AD-520058.1 19.11994 1.596287 25.62188 10.7099 30.55429 4.855674 73.41137 19.43665 AD-1010730.1 21.73395 10.6979 16.70555 2.65559 32.62999 3.77557 56.71824 9.339537 AD-1010731.1 18.79349 9.614942 20.33723 5.175419 21.75738 3.898997 34.06692 1.207849 AD-1010732.1 12.59156 2.240752 15.64331 7.150228 19.65225 1.464936 34.3364 5.246713 AD-520062.8 14.21406 1.676272 15.87543 2.458405 26.63561 8.918796 30.5421 5.165147 AD-520063.4 16.75671 8.284338 23.80043 7.588999 32.82854 12.10351 59.92505 16.52704 AD-1010733.1 22.32816 7.236788 25.92854 23.7787 35.99174 17.74611 60.47482 14.91008 AD-1010734.1 18.24302 1.752058 18.36241 1.316487 23.71195 4.176413 41.71739 6.863728 AD-520066.3 21.0506 1.763814 23.55916 7.45988 27.17446 7.015571 43.35977 15.45225 AD-520067.4 14.92753 1.830658 18.13011 4.290801 35.8336 8.2811 40.55644 8.154041 AD-75289.5 26.44845 8.977692 20.89283 2.747366 34.44447 3.263865 83.95798 24.73915 AD-520068.4 38.03872 12.48769 46.54654 12.13287 35.73126 8.737018 57.92283 4.998898 AD-520069.4 25.91186 7.625084 30.85173 14.5031 31.7561 3.069982 54.3584 6.314143 AD-1010735.1 16.92219 0.846523 35.02702 7.741908 26.23645 1.86282 61.5258 10.92313 AD-67584.6 25.88229 6.362588 22.72034 3.768779 30.82832 0.453279 66.79276 7.638439

Example 6. In Vivo Screening of dsRNA Duplexes in Mice

Duplexes of interest, identified from the above in vitro SAR studies, were evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹⁰ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding a portion of human PNPLA3 mRNA encoding the open reading frame and 3′ UTR of human PNPLA3 mRNA referenced as NM_025225.2, referred to as AAV8-TBG-PI-PNPLA3.

At day 0, groups of three mice were subcutaneously administered a single 10 mg/kg dose of the agents of interest or PBS control. Table 40 provides the treatment groups and Table 41 provides the duplexes of interest. At day 7 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to a housekeeping gene, GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 42 and shown in FIG. 5, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 40 Treatment Groups Treatment Dose Timepoint PBS n/a Day 7 Naïve AD-519345.1 AD-519346.1 AD-519347.1 AD-67554.7 AD-519752.3 AD-1010731.1 AD-1010732.1 AD-519343.1 AD-519344.1 AD-519349.1 AD-519350.1 AD-519753.2 AD-519932.1 AD-519935.2 AD-520018.6 (+CTL)

TABLE 41 Duplexes of Interest DuplexID Range in NM_025225.2 AD-519345.1 1210-1232 AD-519346.1 1211-1233 AD-519347.1 1212-1234 AD-67554.7 1738-1760 AD-519752.3 1739-1761 AD-1010731.1 2110-2132 AD-1010732.1 2111-2133 AD-519343.1 1208-1230 AD-519344.1 1209-1231 AD-519349.1 1214-1236 AD-519350.1 1215-1237 AD-519753.2 1740-1762 AD-519932.1 1920-1942 AD-519935.2 1923-1945 AD-520018.6 (+CTL) 2108-2130

TABLE 42 % Message Duplex Remaining SD PBS Day 7 100.83 7.41 Naïve Day 7 76.79 26.41 AD-519346.1 20.93 7.32 AD-67554.7 24.35 11.53 AD-520018.6 (+CTL) 24.99 1.97 AD-519350.1 25.42 2.79 AD-519347.1 25.76 8.01 AD-519349.1 28.37 10.58 AD-519345.1 29.15 8.28 AD-519753.2 36.95 13.35 AD-519752.3 39.67 5.40 AD-519343.1 39.81 12.85 AD-519932.1 40.63 1.38 AD-1010732.1 42.63 4.42 AD-1010731.1 42.89 5.68 AD-519935.2 50.93 8.55 AD-519344.1 59.71 22.61

Additional duplexes of interest, identified from the above in vitro SAR studies, were evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹⁰ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding the full transcript of NM_025225.2 of human PNPLA3 mRNA, referred to as AAV8-TBG-PI-PNPLA3.

At day 0, groups of three mice were subcutaneously administered a single 10 mg/kg dose of the agents of interest or PBS control. Table 43 provides the treatment groups and Table 44 provides the duplexes of interest. At day 7 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mLRNA levels were compared to a housekeeping gene, GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 45 and shown in FIG. 6, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 43 Treatment Groups Group # Animal # Treatment Dose Timepoint 1 1 PBS n/a D7 2 3 2 4 Naïve n/a 5 6 3 7 AD-517837.2 10 mpk 8 9 4 10 AD-805635.2 11 12 5 13 AD-519329.2 14 15 6 16 AD-520063.2 17 18 7 19 AD-519757.2 20 21 8 22 AD-805631.2 23 24 9 25 AD-516917.2 26 27 10 28 AD-516828.2 29 30 11 31 AD-518983.2 32 33 12 34 AD-805636.2 35 36 13 37 AD-519754.7 38 39 14 40 AD-520062.2 41 42

TABLE 44 Duplexes of Interest DuplexID Range in NM_025225.2 AD-517837.2 1901-1923 AD-805635.2 2182-2200 AD-519329.2 1192-1214 AD-520063.2 2179-2201 AD-519757.2 1744-1766 AD-805631.2 1267-1285 AD-516917.2 795-817 AD-516828.2 677-699 AD-518983.2 773-795 AD-805636.2 1219-1237 AD-519754.7 1741-1763 AD-520062.2 2178-2200

TABLE 45 % Message Duplex Remaining SD PBS Day 7 103.97 40.22 Naïve Day 7 89.23 68.18 AD-517837.2 65.26 22.46 AD-805635.2 57.62 37.80 AD-519329.2 61.36 18.29 AD-520063.2 32.76 9.36 AD-519757.2 22.05 11.75 AD-805631.2 19.04 29.79 AD-516917.2 62.84 35.48 AD-516828.2 88.00 22.18 AD-518983.2 113.79 21.80 AD-805636.2 53.33 6.27 AD-519754.7 70.77 17.96 AD-520062.2 41.73 12.10

Additional duplexes of interest, identified from the above in vitro SAR studies, were evaluated in vivo. In particular, at pre-dose day −14 wild-type mice (C57BL/6) were transduced by intravenous administration of 2×10¹⁰ viral particles of an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3. In particular, mice were administered an AAV8 encoding the open reading frame and 3′ UTR of human PNPLA3 mRNA referenced as NM_025225.2, referred to as AAV8-TBG-PI-PNPLA3.

At day 0, groups of three mice were subcutaneously administered a single 10 mg/kg dose of the agents of interest or PBS control. Table 46 provides the treatment groups and Table 47 provides the duplexes of interest. At day 7 post-dose animals were sacrificed, liver samples were collected and snap-frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by the RT-QPCR method.

Human PNPLA3 mRNA levels were compared to a housekeeping gene, GAPDH. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, listed in Table 48 and shown in FIG. 7, demonstrate that the exemplary duplex agents tested effectively reduce the level of the human PNPLA3 messenger RNA in vivo.

TABLE 46 Treatment Groups Group # Animal # Treatment Dose Timepoint 1 1 PBS n/a D7 2 3 2 4 Naïve 5 6 3 7 AD-67575.9 10 mpk 8 9 4 10 AD-518923.3 11 12 5 13 AD-520053.4 14 15 6 16 AD-519667.2 17 18 7 19 AD-519773.2 20 21 8 22 AD-519354.2 23 24 9 25 AD-520060.4 26 27 10 28 AD-520061.4 29 30 11 31 AD-520062.9 32 33 12 34 AD-520063.5 35 36 13 37 AD-1010733.2 38 39 14 40 AD-1010735.2 41 42 15 43 AD-520018.4 (+CTL) 44 45

TABLE 47 Duplexes of Interest Range in DuplexID NM_025225.2 AD-67575.9 2241-2263 AD-518923.3 683-705 AD-520053.4 2169-2191 AD-519667.2 1631-1653 AD-519773.2 1760-1782 AD-519354.2 1219-1241 AD-520060.4 2176-2198 AD-520061.4 2177-2199 AD-520062.9 2178-2200 AD-520063.5 2179-2201 AD-1010733.2 2114-2136 AD-1010735.2 2121-2143 AD-520018.4 (+CTL) 2108-2130

TABLE 48 % Message Duplex Remaining SD PBS Day 7 101.90 6.24 Naïve Day 7 78.59 3.28 AD-519773.2 26.03 3.77 AD-520018.4 33.95 10.54 (+CTL) AD-520053.4 34.87 3.84 AD-520063.5 39.46 2.44 AD-520062.9 43.73 13.15 AD-1010733.2 45.97 8.41 AD-520060.4 49.04 16.64 AD-520061.4 49.32 4.29 AD-1010735.2 50.04 10.92 AD-519354.2 50.21 22.03 AD-519667.2 51.76 10.29 AD-67575.9 63.91 15.29 AD-518923.3 143.13 33.05

Example 7. Design, Synthesis and In Vitro Screening of Additional dsRNA Duplexes

Additional siRNAs were designed, synthesized and prepared using methods known in the art and described above in Example 1.

Detailed lists of the additional unmodified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 49. Detailed lists of the modified PNPLA3 sense and antisense strand nucleotide sequences are shown in Table 50.

Single dose screens of the additional agents were performed by free uptake and transfection.

For free uptake, experiments were performed by adding 2.5 μl of siRNA duplexes in PBS per well into a 96 well plate. Complete growth media (47.5 μl) containing about 1.5×10⁴ primary cynomolgus hepatocytes (PCH) were then added to the siRNA. Cells were incubated for 48 hours prior to RNA purification and RT-qPCR. Single dose experiments were performed at 500 nM, 100 nM, 10, and 1 nM final duplex concentration.

For transfection, Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 7.5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 2.5 μl of each siRNA duplex to an individual well in a 384-well plate. The mixture was then incubated at room temperature for 15 minutes. Forty μl of complete growth media without antibiotic containing ˜1.5×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM final duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 101 of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 3 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10× dNTPs, 1.5 μl Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNase inhibitor and 9.9 μl of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37° C. for 2 hours. Following this, the plates were agitated at 80° C. for 8 minutes.

RT-qPCR was performed as described above and relative fold change was calculated as described above.

The results of the transfection assays of the dsRNA agents listed in Tables 49 and 50 in Hep3B cells are shown in Table 51. The results of the free uptake experiment of the dsRNA agents listed in Tables 49 and 50 in primary cynomolgus hepatocytes (PCH) are shown in Table 52.

TABLE 49 Unmodified Sense and Antisense Strand Sequencesof PNPLA3 dsRNA Agents SEQ SEQ ID Range in Antisense ID Range in Duplex Name Sense Sequence 5′ to 3′ NO: NM_025225.2 Sequence 5′ to 3′ NO: NM_025225.2 AD-67554.9 UCUGAGCUGAGUUGGUUUUAU 2894 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3074 1738-1760 AD-1193317.1 UCUGAGCUGAGUUGGUUUUAU 2895 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3075 1738-1760 AD-1193318.1 UCUGAGCUGAGUUGGUUUUAU 2896 1740-1760 ATAAAACCAACTCAGCUCAGAGG 3076 1738-1760 AD-1193319.1 UCUGAGCUGAGUUGGUUUUAU 2897 1740-1760 AUAAAACCAACUCAGCUCAGACC 3077 1738-1760 AD-1193320.1 UCUGAGCUGAGUUGGUUUUAU 2898 1740-1760 ATAAAACCAACTCAGCUCAGACC 3078 1738-1760 AD-1193321.1 UGAGCUGAGUUGGUUUUAU 2899 1742-1760 AUAAAACCAACUCAGCUCAGC 3079 1740-1760 AD-1193322.1 UGAGCUGAGUUGGUUUUAU 2900 1742-1760 ATAAAACCAACTCAGCUCAGC 3080 1740-1760 AD-1193344.1 UCUGAGCUGAGUUGGUUUUAU 2901 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3081 1738-1760 AD-1193324.1 UCUGAGCUGAGUUGGUUUUAU 2902 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3082 1738-1760 AD-1193325.1 UCUGAGCUGAGUUGGUUUUAU 2903 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3083 1738-1760 AD-1193326.1 UCUGAGCUGAGUUGGUUUUAU 2904 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3084 1738-1760 AD-1193327.1 UCUGAGCUGAGUUGGUUUUAU 2905 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3085 1738-1760 AD-1193328.1 UCUGAGCUGAGUUGGUUUUAU 2906 1740-1760 AUAAAACCAACUCAGCUCAGAGG 3086 1738-1760 AD-1193329.1 UCUGAGCUGAGUUGGUUUUAU 2907 1740-1760 AUAAAACCAACUCAGCUCAGACC 3087 1738-1760 AD-1193330.1 UCUGAGCUGAGUUGGUUUUAU 2908 1740-1760 AUAAAACCAACUCAGCUCAGACC 3088 1738-1760 AD-1193331.1 UCUGAGCUGAGUUGGUUUUAU 2909 1740-1760 AUAAAACCAACUCAGCUCAGACC 3089 1738-1760 AD-1193332.1 UGAGCUGAGUUGGUUUUAU 2910 1742-1760 AUAAAACCAACUCAGCUCAGC 3090 1740-1760 AD-1193333.1 UGAGCUGAGUUGGUUUUAU 2911 1742-1760 AUAAAACCAACUCAGCUCAGC 3091 1740-1760 AD-1193334.1 UGAGCUGAGUUGGUUUUAU 2912 1742-1760 AUAAAACCAACUCAGCUCAGC 3092 1740-1760 AD-1193335.1 UCUGAGCUGAGUUGGUUUUAU 2913 1740-1760 ATAAAACCAACTCAGCUCAGAGG 3093 1738-1760 AD-1193336.1 UCUGAGCUGAGUUGGUTUUAU 2914 1740-1760 ATAAAACCAACTCAGCUCAGAGG 3094 1738-1760 AD-519346.3 CCAUUAGGAUAAUGUCUUAUU 2915 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3095 1211-1233 AD-1193337.1 CCAUUAGGAUAAUGUCUUAUU 2916 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3096 1211-1233 AD-1193338.1 CCAUUAGGAUAAUGUCUUAUU 2917 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3097 1211-1233 AD-1193339.1 CCAUUAGGAUAAUGUCUUAUU 2918 1213-1233 AAUAAGACAUUAUCCUAAUGGCC 3098 1211-1233 AD-1193340.1 CCAUUAGGAUAAUGUCUUAUU 2919 1213-1233 AAUAAGACAUUAUCCUAAUGGCC 3099 1211-1233 AD-1193341.1 CCAUUAGGAUAAUGUCUUAUU 2920 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3100 1211-1233 AD-1193342.1 CCAUUAGGAUAAUGUCUUAUU 2921 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3101 1211-1233 AD-1193343.1 CCAUUAGGAUAAUGUCUUAUU 2922 1213-1233 AAUAAGACAUUAUCCUAAUGGCC 3102 1211-1233 AD-1193344.1 CCAUUAGGAUAAUGUCUUAUU 2923 1213-1233 AAUAAGACAUUAUCCUAAUGGCC 3103 1211-1233 AD-1193345.1 AUUAGGAUAAUGUCUUAUU 2924 1215-1233 AAUAAGACAUUAUCCUAAUGG 3104 1213-1233 AD-1193346.1 AUUAGGAUAAUGUCUUAUU 2925 1215-1233 AAUAAGACAUUAUCCUAAUGG 3105 1213-1233 AD-1193347.1 CCAUUAGGAUAAUGUCUUAUU 2926 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3106 1211-1233 AD-1193348.1 CCAUUAGGAUAAUGUCUUAUU 2927 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3107 1211-1233 AD-1193349.1 CCAUUAGGAUAAUGUCTUAUU 2928 1213-1233 AAUAAGACAUUAUCCUAAUGGGU 3108 1211-1233 AD-519347.4 CAUUAGGAUAAUGUCUUAUGU 2929 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3109 1212-1234 AD-1193350.1 CAUUAGGAUAAUGUCUUAUGU 2930 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3110 1212-1234 AD-1193351.1 CAUUAGGAUAAUGUCUUAUGU 2931 1214-1234 ACAUAAGACAUUAUCCUAAUGCC 3111 1212-1234 AD-1193352.1 CAUUAGGAUAAUGUCUUAUGU 2932 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3112 1212-1234 AD-1193353.1 CAUUAGGAUAAUGUCUUAUGU 2933 1214-1234 ACAUAAGACAUUAUCCUAAUGCC 3113 1212-1234 AD-1193354.1 UUAGGAUAAUGUCUUAUGU 2934 1216-1234 ACAUAAGACAUUAUCCUAAUG 3114 1214-1234 AD-1193355.1 CAUUAGGATAAUGUCUUAUGU 2935 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3115 1212-1234 AD-1193356.1 CAUUAGGATAAUGUCUTAUGU 2936 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3116 1212-1234 AD-1193357.1 CAUUAGGAUAAUGUCUUAUGU 2937 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3117 1212-1234 AD-1193358.1 CAUUAGGAUAAUGUCUTAUGU 2938 1214-1234 ACAUAAGACAUUAUCCUAAUGGG 3118 1212-1234 AD-519350.4 UAGGAUAAUGUCUUAUGUAAU 2939 1217-1237 AUUACAUAAGACAUUAUCCUAAU 3119 1215-1237 AD-1193359.1 UAGGAUAAUGUCUUAUGUAAU 2940 1217-1237 AUUACATAAGACAUUAUCCUAAU 3120 1215-1237 AD-1193360.1 UAGGAUAAUGUCUUAUGUAAU 2941 1217-1237 AUUACATAAGACAUUAUCCUAAU 3121 1215-1237 AD-1193361.1 UAGGAUAAUGUCUUAUGUAAU 2942 1217-1237 AUUACATAAGACAUUAUCCUAGU 3122 1215-1237 AD-1193362.1 UAGGAUAAUGUCUUAUGUAAU 2943 1217-1237 AUUACATAAGACAUUAUCCUAGU 3123 1215-1237 AD-1193363.1 UAGGAUAAUGUCUUAUGUAAU 2944 1217-1237 AUUACATAAGACAUUAUCCUACC 3124 1215-1237 AD-1193364.1 UAGGAUAAUGUCUUAUGUAAU 2945 1217-1237 AUUACATAAGACAUUAUCCUACC 3125 1215-1237 AD-1193365.1 UAGGAUAAUGUCUUAUGUAAU 2946 1217-1237 AUUACATAAGACAUUAUCCUAGU 3126 1215-1237 AD-1193366.1 UAGGAUAAUGUCUUAUGUAAU 2947 1217-1237 AUUACATAAGACAUUAUCCUAGU 3127 1215-1237 AD-1193367.1 UAGGAUAAUGUCUUAUGUAAU 2948 1217-1237 AUUACATAAGACAUUAUCCUAGU 3128 1215-1237 AD-1193368.1 UAGGAUAAUGUCUUAUGUAAU 2949 1217-1237 AUUACATAAGACAUUAUCCUAGU 3129 1215-1237 AD-1193369.1 UAGGAUAAUGUCUUAUGUAAU 2950 1217-1237 AUUACATAAGACAUUAUCCUAGU 3130 1215-1237 AD-1193370.1 UAGGAUAAUGUCUUAUGUAAU 2951 1217-1237 AUUACATAAGACAUUAUCCUAGU 3131 1215-1237 AD-1193371.1 UAGGAUAAUGUCUUAUGUAAU 2952 1217-1237 AUUACATAAGACAUUAUCCUAGU 3132 1215-1237 AD-1193372.1 UAGGAUAAUGUCUUAUGUAAU 2953 1217-1237 AUUACATAAGACAUUAUCCUAGU 3133 1215-1237 AD-519757.4 CUGAGUUGGUUUUAUGAAAAU 2954 1746-1766 AUUUUCAUAAAACCAACUCAGCU 3134 1744-1766 AD-1193373.1 CUGAGUUGGUUUUAUGAAAAU 2955 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3135 1744-1766 AD-1193374.1 CUGAGUUGGUUUUAUGAAAAU 2956 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3136 1744-1766 AD-1193375.1 CUGAGUUGGUUUUAUGAAAAU 2957 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3137 1744-1766 AD-1193376.1 CUGAGUUGGUUUUAUGAAAAU 2958 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3138 1744-1766 AD-1193377.1 CUGAGUUGGUUUUAUGAAAAU 2959 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3139 1744-1766 AD-1193378.1 CUGAGUUGGUUUUAUGAAAAU 2960 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3140 1744-1766 AD-1193379.1 CUGAGUUGGUUUUAUGAAAAU 2961 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3141 1744-1766 AD-1193380.1 CUGAGUUGGUUUUAUGAAAAU 2962 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3142 1744-1766 AD-1193381.1 CUGAGUUGGUUUUAUGAAAAU 2963 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3143 1744-1766 AD-1193382.1 CUGAGUUGGUUUUAUGAAAAU 2964 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3144 1744-1766 AD-1193383.1 GAGUUGGUUUUAUGAAAAU 2965 1748-1766 AUUUTCAUAAAACCAACUCGG 3145 1746-1766 AD-1193384.1 GAGUUGGUUUUAUGAAAAU 2966 1748-1766 AUUUTCAUAAAACCAACUCGG 3146 1746-1766 AD-1193385.1 CUGAGUUGGUUUUAUGAAAAU 2967 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3147 1744-1766 AD-1193386.1 CUGAGUUGGUUUUAUGAAAAU 2968 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3148 1744-1766 AD-1193387.1 CUGAGUUGGUUUUAUGAAAAU 2969 1746-1766 AUUUTCAUAAAACCAACUCAGCU 3149 1744-1766 AD-1193388.1 CUGAGUUGGUUUUAUGAAAAU 2970 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3150 1744-1766 AD-1193389.1 CUGAGUUGGUUUUAUGAAAAU 2971 1746-1766 AUUUTCAUAAAACCAACUCAGUC 3151 1744-1766 AD-1193390.1 GAGUUGGUUUUAUGAAAAU 2972 1748-1766 AUUUTCAUAAAACCAACUCGG 3152 1746-1766 AD-1193391.1 GAGUUGGUUUUAUGAAAAU 2973 1748-1766 AUUUTCAUAAAACCAACUCGG 3153 1746-1766 AD-520018.9 GAUAACCUUGACUACUAAAAU 2974 2110-2130 AUUUUAGUAGUCAAGGUUAUCAU 3154 2108-2130 AD-1193392.1 GAUAACCUUGACUACUAAAAU 2975 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3155 2108-2130 AD-1193393.1 GAUAACCUUGACUACUAAAAU 2976 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3156 2108-2130 AD-1193394.1 GAUAACCUUGACUACUAAAAU 2977 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3157 2108-2130 AD-1193395.1 GAUAACCUUGACUACUAAAAU 2978 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3158 2108-2130 AD-1193396.1 GAUAACCUUGACUACUAAAAU 2979 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3159 2108-2130 AD-1193397.1 GAUAACCUUGACUACUAAAAU 2980 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3160 2108-2130 AD-1193398.1 GAUAACCUUGACUACUAAAAU 2981 2110-2130 AUUUUAGUAGUCAAGGUUAUCAU 3161 2108-2130 AD-1193399.1 GAUAACCUUGACUACUAAAAU 2982 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3162 2108-2130 AD-1193400.1 GAUAACCUUGACUACUAAAAU 2983 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3163 2108-2130 AD-1193401.1 GAUAACCUUGACUACUAAAAU 2984 2110-2130 AUUUTAGUAGUCAAGGUUAUCAU 3164 2108-2130 AD-1193402.1 GAUAACCUUGACUACUAAAAU 2985 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3165 2108-2130 AD-1193403.1 GAUAACCUUGACUACUAAAAU 2986 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3166 2108-2130 AD-1193404.1 GAUAACCUUGACUACUAAAAU 2987 2110-2130 AUUUTAGUAGUCAAGGUUAUCGU 3167 2108-2130 AD-1193405.1 UAACCUUGACUACUAAAAU 2988 2112-2130 AUUUTAGUAGUCAAGGUUAUC 3168 2110-2130 AD-1193406.1 UAACCUUGACUACUAAAAU 2989 2112-2130 AUUUTAGUAGUCAAGGUUAUC 3169 2110-2130 AD-1193407.1 UAACCUUGACUACUAAAAU 2990 2112-2130 AUUUTAGUAGUCAAGGUUAUC 3170 2110-2130 AD-520053.5 UUUUAGAACACCUUUUUCACU 2991 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3171 2169-2191 AD-1193408.1 UUUUAGAACACCUUUUUCACU 2992 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3172 2169-2191 AD-1193409.1 UUUUAGAACACCUUUUUCACU 2993 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3173 2169-2191 AD-1193410.1 UUUUAGAACACCUUUUUCACU 2994 2171-2191 AGUGAAAAAGGTGUUCUAAAAUU 3174 2169-2191 AD-1193411.1 UUUUAGAACACCUUUUUCACU 2995 2171-2191 AGUGAAAAAGGUGUUCUAAAACC 3175 2169-2191 AD-1193412.1 UUUUAGAACACCUUUUUCACU 2996 2171-2191 AGUGAAAAAGGUGUUCUAAAACC 3176 2169-2191 AD-1193413.1 UUUUAGAACACCUUUUUCACU 2997 2171-2191 AGUGAAAAAGGTGUUCUAAAACC 3177 2169-2191 AD-1193414.1 UUUUAGAACACCUUUUUCACU 2998 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3178 2169-2191 AD-1193415.1 UUUUAGAACACCUUUUUCACU 2999 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3179 2169-2191 AD-1193416.1 UUUUAGAACACCUUUUUCACU 3000 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3180 2169-2191 AD-1193417.1 UUUUAGAACACCUUUUUCACU 3001 2171-2191 AGUGAAAAAGGTGUUCUAAAAUU 3181 2169-2191 AD-1193418.1 UUUUAGAACACCUUUUUCACU 3002 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3182 2169-2191 AD-1193419.1 UUUUAGAACACCUUUUUCACU 3003 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3183 2169-2191 AD-1193420.1 UUUUAGAACACCUUUUUCACU 3004 2171-2191 AGUGAAAAAGGUGUUCUAAAAUU 3184 2169-2191 AD-1193421.1 UUUUAGAACACCUUUUUCACU 3005 2171-2191 AGUGAAAAAGGTGUUCUAAAAUU 3185 2169-2191 AD-1193422.1 GUGGAAUCUGCCAUUGCGA 3006 1257-1275 UCGCAATGGCAGAUUCCACAG 3186 1255-1275 AD-1193423.1 GUGGAAUCUGCCAUUGCGA 3007 1257-1275 UCGCAATGGCAGAUUCCACGG 3187 1255-1275 AD-1193424.1 UGGAAUCUGCCAUUGCGAU 3008 1258-1276 AUCGCAAUGGCAGAUUCCACA 3188 1256-1276 AD-1193425.1 UGGAAUCUGCCAUUGCGAU 3009 1258-1276 AUCGCAAUGGCAGAUUCCACG 3189 1256-1276 AD-1193426.1 GGAAUCUGCCAUUGCGAUU 3010 1259-1277 AAUCGCAAUGGCAGAUUCCAC 3190 1257-1277 AD-1193427.1 GGAAUCUGCCAUUGCGAUU 3011 1259-1277 AAUCGCAAUGGCAGAUUCCGC 3191 1257-1277 AD-1193428.1 GAAUCUGCCAUUGCGAUUU 3012 1260-1278 AAAUCGCAAUGGCAGAUUCCA 3192 1258-1278 AD-1193429.1 GAAUCUGCCAUUGCGAUUU 3013 1260-1278 AAAUCGCAAUGGCAGAUUCCG 3193 1258-1278 AD-1193430.1 AAUCUGCCAUUGCGAUUGU 3014 1261-1279 ACAATCGCAAUGGCAGAUUCC 3194 1259-1279 AD-1193431.1 AAUCUGCCAUUGCGAUUGU 3015 1261-1279 ACAATCGCAAUGGCAGAUUCC 3195 1259-1279 AD-1193432.1 AUCUGCCAUUGCGAUUGUU 3016 1262-1280 AACAAUCGCAAUGGCAGAUUC 3196 1260-1280 AD-1193433.1 AUCUGCCAUUGCGAUUGUU 3017 1262-1280 AACAAUCGCAATGGCAGAUUC 3197 1260-1280 AD-1193434.1 UCUGCCAUUGCGAUUGUCU 3018 1263-1281 AGACAATCGCAAUGGCAGAUU 3198 1261-1281 AD-1193435.1 UCUGCCAUUGCGAUUGUCU 3019 1263-1281 AGACAATCGCAAUGGCAGAUU 3199 1261-1281 AD-1193436.1 CUGCCAUUGCGAUUGUCCA 3020 1264-1282 UGGACAAUCGCAAUGGCAGAU 3200 1262-1282 AD-1193437.1 CUGCCAUUGCGAUUGUCCA 3021 1264-1282 UGGACAAUCGCAAUGGCAGAU 3201 1262-1282 AD-1193438.1 UGCCAUUGCGAUUGUCCAU 3022 1265-1283 AUGGACAAUCGCAAUGGCAGA 3202 1263-1283 AD-1193439.1 UGCCAUUGCGAUUGUCCAU 3023 1265-1283 AUGGACAAUCGCAAUGGCAGG 3203 1263-1283 AD-1193440.1 GCCAUUGCGAUUGUCCAGA 3024 1266-1284 UCUGGACAAUCGCAAUGGCGG 3204 1264-1284 AD-805631.3 CCAUUGCGAUUGUCCAGAU 3025 1267-1285 AUCUGGACAAUCGCAAUGG 3205 1267-1285 AD-1193441.1 CCAUUGCGAUUGUCCAGAU 3026 1267-1285 AUCUGGACAAUCGCAAUGG 3206 1267-1285 AD-1193442.1 CCAUUGCGAUUGUCCAGAU 3027 1267-1285 AUCUGGACAAUCGCAAUGGCA 3207 1265-1285 AD-1193443.1 CCAUUGCGAUUGUCCAGAU 3028 1267-1285 AUCUGGACAAUCGCAAUGGCG 3208 1265-1285 AD-1193444.1 CAUUGCGAUUGUCCAGAGA 3029 1268-1286 UCUCTGGACAAUCGCAAUGGC 3209 1266-1286 AD-1193445.1 AUUGCGAUUGUCCAGAGAU 3030 1269-1287 AUCUCUGGACAAUCGCAAUGG 3210 1267-1287 AD-1193446.1 CAUUGCGAUUGUCCAGAGACU 3031 1268-1288 AGUCTCTGGACAAUCGCAAUGGC 3211 1266-1288 AD-1193447.1 UGCGAUUGUCCAGAGACUU 3032 1271-1289 AAGUCUCUGGACAAUCGCAGU 3212 1269-1289 AD-1193448.1 GCGAUUGUCCAGAGACUGU 3033 1272-1290 ACAGTCTCUGGACAAUCGCAA 3213 1270-1290 AD-1193449.1 UUGCGAUUGUCCAGAGACUGU 3034 1270-1290 ACAGTCTCUGGACAAUCGCAAUG 3214 1268-1290 AD-1193450.1 CGAUUGUCCAGAGACUGGU 3035 1273-1291 ACCAGUCUCUGGACAAUCGCA 3215 1271-1291 AD-1193451.1 CGAUUGUCCAGAGACUGGU 3036 1273-1291 ACCAGUCUCUGGACAAUCGCG 3216 1271-1291 AD-1193452.1 GAUUGUCCAGAGACUGGUU 3037 1274-1292 AACCAGTCUCUGGACAAUCGC 3217 1272-1292 AD-1193452.2 GAUUGUCCAGAGACUGGUU 3038 1274-1292 AACCAGTCUCUGGACAAUCGC 3218 1272-1292 AD-1193453.1 AUUGUCCAGAGACUGGUGA 3039 1275-1293 UCACCAGUCUCUGGACAAUCG 3219 1273-1293 AD-1193454.1 AUUGUCCAGAGACUGGUGA 3040 1275-1293 UCACCAGUCUCTGGACAAUCG 3220 1273-1293 AD-1193455.1 UUGUCCAGAGACUGGUGAU 3041 1276-1294 AUCACCAGUCUCUGGACAAUC 3221 1274-1294 AD-1193456.1 UUGUCCAGAGACUGGUGAU 3042 1276-1294 AUCACCAGUCUCUGGACAAUC 3222 1274-1294 AD-1193457.1 UGUCCAGAGACUGGUGACA 3043 1277-1295 UGUCACCAGUCTCUGGACAGU 3223 1275-1295 AD-1193458.1 UGGUUUUAUGAAAAGCUAGGA 3044 1752-1772 UCCUAGCUUUUCAUAAAACCAAC 3224 1750-1772 AD-1193459.1 UGGUUUUAUGAAAAGCUAGGA 3045 1752-1772 UCCUAGCUUUUCAUAAAACCAGC 3225 1750-1772 AD-1193460.1 GGUUUUAUGAAAAGCUAGGAA 3046 1753-1773 UUCCTAGCUUUUCAUAAAACCGG 3226 1751-1773 AD-1193461.1 GUUUUAUGAAAAGCUAGGAAU 3047 1754-1774 AUUCCUAGCUUUUCAUAAAACCA 3227 1752-1774 AD-1193462.1 GUUUUAUGAAAAGCUAGGAAU 3048 1754-1774 AUUCCUAGCUUUUCAUAAAACCG 3228 1752-1774 AD-1193463.1 UUUUAUGAAAAGCUAGGAAGU 3049 1755-1775 ACUUCCTAGCUUUUCAUAAAACC 3229 1753-1775 AD-1193464.1 UUUAUGAAAAGCUAGGAAGCA 3050 1756-1776 UGCUTCCUAGCUUUUCAUAAAGC 3230 1754-1776 AD-1193465.1 UUAUGAAAAGCUAGGAAGCAA 3051 1757-1777 UUGCTUCCUAGCUUUUCAUAAGG 3231 1755-1777 AD-1193466.1 UAUGAAAAGCUAGGAAGCAAU 3052 1758-1778 AUUGCUTCCUAGCUUUUCAUAGG 3232 1756-1778 AD-1193467.1 AUGAAAAGCUAGGAAGCAACU 3053 1759-1779 AGUUGCTUCCUAGCUUUUCAUGG 3233 1757-1779 AD-1193468.1 UGAAAAGCTAGGAAGCAACCU 3054 1760-1780 AGGUTGCUUCCTAGCUUUUCAUG 3234 1758-1780 AD-1193469.1 GAAAAGCUAGGAAGCAACCUU 3055 1761-1781 AAGGTUGCUUCCUAGCUUUUCGU 3235 1759-1781 AD-519773.3 AAAAGCUAGGAAGCAACCUUU 3056 1762-1782 AAAGGUUGCUUCCUAGCUUUUCA 3236 1760-1782 AD-1193470.1 AAAAGCUAGGAAGCAACCUUU 3057 1762-1782 AAAGGUTGCUUCCUAGCUUUUCA 3237 1760-1782 AD-1193471.1 AAAAGCUAGGAAGCAACCUUU 3058 1762-1782 AAAGGUTGCUUCCUAGCUUUUCG 3238 1760-1782 AD-1193472.1 AAAGCUAGGAAGCAACCUUUU 3059 1763-1783 AAAAGGTUGCUUCCUAGCUUUUC 3239 1761-1783 AD-1193473.1 AAGCUAGGAAGCAACCUUUCU 3060 1764-1784 AGAAAGGUUGCUUCCUAGCUUUU 3240 1762-1784 AD-1193474.1 AGCUAGGAAGCAACCUUUCGU 3061 1765-1785 ACGAAAGGUUGCUUCCUAGCUUU 3241 1763-1785 AD-1193475.1 CUAGGAAGCAACCUUUCGU 3062 1767-1785 ACGAAAGGUUGCUUCCUAGCU 3242 1765-1785 AD-1193476.1 GCUAGGAAGCAACCUUUCGCU 3063 1766-1786 AGCGAAAGGUUGCUUCCUAGCUU 3243 1764-1786 AD-1193477.1 UAGGAAGCAACCUUUCGCU 3064 1768-1786 AGCGAAAGGUUGCUUCCUAGC 3244 1766-1786 AD-1193478.1 CUAGGAAGCAACCUUUCGCCU 3065 1767-1787 AGGCGAAAGGUUGCUUCCUAGCU 3245 1765-1787 AD-1193479.1 AGGAAGCAACCUUUCGCCU 3066 1769-1787 AGGCGAAAGGUUGCUUCCUGG 3246 1767-1787 AD-1193480.1 UAGGAAGCAACCUUUCGCCUU 3067 1768-1788 AAGGCGAAAGGUUGCUUCCUAGC 3247 1766-1788 AD-1193481.1 GGAAGCAACCUUUCGCCUU 3068 1770-1788 AAGGCGAAAGGTUGCUUCCUG 3248 1768-1788 AD-1193482.1 AGGAAGCAACCUUUCGCCUGU 3069 1769-1789 ACAGGCGAAAGGUUGCUUCCUAG 3249 1767-1789 AD-1193483.1 GAAGCAACCUUUCGCCUGU 3070 1771-1789 ACAGGCGAAAGGUUGCUUCCU 3250 1769-1789 AD-1193484.1 AAGCAACCUUUCGCCUGUU 3071 1772-1790 AACAGGCGAAAGGUUGCUUCC 3251 1770-1790 AD-1193485.1 AGCAACCUUUCGCCUGUGU 3072 1773-1791 ACACAGGCGAAAGGUUGCUUC 3252 1771-1791 AD-1193486.1 GCAACCUUUCGCCUGUGCA 3073 1774-1792 UGCACAGGCGAAAGGUUGCUU 3253 1772-1792

TABLE 50 Modified Sense and Antisense Strand Sequencesof PNPLA3 dsRNA Agents Duplex Name Sense Sequence 5′ to 3′ SEQ ID NO: Antisense Sequence 5′ to 3′ SEQ ID NO: mRNA Target Sequence SEQ ID NO: AD-67554.9 uscsugagCfuGfAfGfuugguuuuauL96 3254 asUfsaaaAfcCfAfacucAfgCfucagasgsg 3434 CCUCUGAGCUGAGUUGGUUUUAU 3614 AD-1193317.1 uscsugagCfuGfAfGfuugguuuuauL96 3255 asUfsaadAadCcaacucAfgCfucagasgsg 3435 CCUCUGAGCUGAGUUGGUUUUAU 3615 AD-1193318.1 uscsugagCfuGfAfGfuugguuuuauL96 3256 asdTsaadAadCcaacdTcdAgdCucagasgsg 3436 CCUCUGAGCUGAGUUGGUUUUAU 3616 AD-1193319.1 uscsugagCfuGfAfGfuugguuuuauL96 3257 asUfsaadAadCcaacucAfgCfucagascsc 3437 CCUCUGAGCUGAGUUGGUUUUAU 3617 AD-1193320.1 uscsugagCfuGfAfGfuugguuuuauL96 3258 asdTsaadAadCcaacdTcdAgdCucagascsc 3438 CCUCUGAGCUGAGUUGGUUUUAU 3618 AD-1193321.1 usgsagCfuGfAfGfuugguuuuauL96 3259 asUfsaadAadCcaacucAfgCfucasgsc 3439 UCUGAGCUGAGUUGGUUUUAU 3619 AD-1193322.1 usgsagCfuGfAfGfuugguuuuauL96 3260 asdTsaadAadCcaacdTcdAgdCucasgsc 3440 UCUGAGCUGAGUUGGUUUUAU 3620 AD-1193323.1 uscsugagCfudGadGuugguuuuauL96 3261 asUfsaaaAfcCfAfacucAfgCfucagasgsg 3441 CCUCUGAGCUGAGUUGGUUUUAU 3621 AD-1193324.1 uscsugagCfugAfdGuugguuuuauL96 3262 asUfsaaaAfcCfAfacucAfgCfucagasgsg 3442 CCUCUGAGCUGAGUUGGUUUUAU 3622 AD-1193325.1 uscsugagCfugadGUfugguuuuauL96 3263 asUfsaaaAfcCfAfacucAfgCfucagasgsg 3443 CCUCUGAGCUGAGUUGGUUUUAU 3623 AD-1193326.1 uscsugagCfudGadGuugguuuuauL96 3264 asUfsaadAadCcaacucAfgCfucagasgsg 3444 CCUCUGAGCUGAGUUGGUUUUAU 3624 AD-1193327.1 uscsugagCfugAfdGuugguuuuauL96 3265 asUfsaadAadCcaacucAfgCfucagasgsg 3445 CCUCUGAGCUGAGUUGGUUUUAU 3625 AD-1193328.1 uscsugagCfugadGUfugguuuuauL96 3266 asUfsaadAadCcaacucAfgCfucagasgsg 3446 CCUCUGAGCUGAGUUGGUUUUAU 3626 AD-1193329.1 uscsugagCfudGadGuugguuuuauL96 3267 asUfsaadAadCcaacucAfgCfucagascsc 3447 CCUCUGAGCUGAGUUGGUUUUAU 3627 AD-1193330.1 uscsugagCfugAfdGuugguuuuauL96 3268 asUfsaadAadCcaacucAfgCfucagascsc 3448 CCUCUGAGCUGAGUUGGUUUUAU 3628 AD-1193331.1 uscsugagCfugadGUfugguuuuauL96 3269 asUfsaadAadCcaacucAfgCfucagascsc 3449 CCUCUGAGCUGAGUUGGUUUUAU 3629 AD-1193332.1 usgsagCfudGadGuugguuuuauL96 3270 asUfsaadAadCcaacucAfgCfucasgsc 3450 UCUGAGCUGAGUUGGUUUUAU 3630 AD-1193333.1 usgsagCfugAfdGuugguuuuauL96 3271 asUfsaadAadCcaacucAfgCfucasgsc 3451 UCUGAGCUGAGUUGGUUUUAU 3631 AD-1193334.1 usgsagCfugadGUfugguuuuauL96 3272 asUfsaadAadCcaacucAfgCfucasgsc 3452 UCUGAGCUGAGUUGGUUUUAU 3632 AD-1193335.1 uscsugagcudGadGuugguuuuauL96 3273 asdTsaadAadCcaacdTcdAgdCucagasgsg 3453 CCUCUGAGCUGAGUUGGUUUUAU 3633 AD-1193336.1 uscsugagcudGadGuuggudTuuauL96 3274 asdTsaaaadCcaacdTcdAgdCucagasgsg 3454 CCUCUGAGCUGAGUUGGUUUUAU 3634 AD-519346.3 cscsauuaGfgAfUfAfaugucuuauuL96 3275 asAfsuaaGfaCfAfuuauCfcUfaauggsgsu 3455 ACCCAUUAGGAUAAUGUCUUAUG 3635 AD-1193337.1 cscsauuaGfgAfUfAfaugucuuauuL96 3276 asAfsuadAgdAcauuauCfcUfaauggsgsu 3456 ACCCAUUAGGAUAAUGUCUUAUG 3636 AD-1193338.1 cscsauuaGfgAfUfAfaugucuuauuL96 3277 asdAsuadAgdAcauuauCfcUfaauggsgsu 3457 ACCCAUUAGGAUAAUGUCUUAUG 3637 AD-1193339.1 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AD-1193428.1 gsasaucuGfCfCfauugcgauuuL96 3372 asAfsaudCgdCaauggcAfgAfuucscsa 3552 UGGAAUCUGCCAUUGCGAUUG 3732 AD-1193429.1 gsasaucugCfCfauugcgauuuL96 3373 asAfsaudCgdCaauggcAfgAfuucscsg 3553 UGGAAUCUGCCAUUGCGAUUG 3733 AD-1193430.1 asasucugCfCfAfuugcgauuguL96 3374 asCfsaadTcdGcaauggCfaGfauuscsc 3554 GGAAUCUGCCAUUGCGAUUGU 3734 AD-1193431.1 asasucugCfCfAfuugcgauuguL96 3375 asCfsaadTcdGcaauggCfagauuscsc 3555 GGAAUCUGCCAUUGCGAUUGU 3735 AD-1193432.1 asuscugcCfAfUfugcgauuguuL96 3376 asAfscadAudCgcaaugGfcAfgaususc 3556 GAAUCUGCCAUUGCGAUUGUC 3736 AD-1193433.1 asuscugcCfAfUfugcgauuguuL96 3377 asAfscadAudCgcaadTgdGcAfgaususc 3557 GAAUCUGCCAUUGCGAUUGUC 3737 AD-1193434.1 uscsugccAfUfUfgcgauugucuL96 3378 asGfsacdAadTcgcaauGfgCfagasusu 3558 AAUCUGCCAUUGCGAUUGUCC 3738 AD-1193435.1 uscsugccAfUfUfgcgauugucuL96 3379 asdGsacdAadTcgcadAudGgCfagasusu 3559 AAUCUGCCAUUGCGAUUGUCC 3739 AD-1193436.1 csusgccaUfUfGfcgauuguccaL96 3380 usGfsgadCadAucgcaaUfgGfcagsasu 3560 AUCUGCCAUUGCGAUUGUCCA 3740 AD-1193437.1 csusgccaUfudGCfgauuguccaL96 3381 usdGsgadCadAucgcaaUfgdGcagsasu 3561 AUCUGCCAUUGCGAUUGUCCA 3741 AD-1193438.1 usgsccauUfGfCfgauuguccauL96 3382 asUfsggdAcdAaucgcaAfuGfgcasgsa 3562 UCUGCCAUUGCGAUUGUCCAG 3742 AD-1193439.1 usgsccauUfgCfgauuguccauL96 3383 asUfsggdAcdAaucgdCaAfudGgcasgsg 3563 UCUGCCAUUGCGAUUGUCCAG 3743 AD-1193440.1 gscscauudGcdGauuguccagaL96 3384 usCfsugdGa(Cgn)aaucgcAfaUfggcsgsg 3564 CUGCCAUUGCGAUUGUCCAGA 3744 AD-805631.3 cscsauugCfgAfUfUfguccagauL96 3385 asUfscugGfacaaucgCfaAfugg 3565 CCAUUGCGAUUGUCCAGAG 3745 AD-1193441.1 cscsauugCfgAfUfUfguccagauL96 3386 asUfscudGg(Agn)caaucgCfaAfusgsg 3566 CCAUUGCGAUUGUCCAGAG 3746 AD-1193442.1 cscsauugCfGfAfuuguccagauL96 3387 asUfscudGg(Agn)caaucgCfaAfuggscsa 3567 UGCCAUUGCGAUUGUCCAGAG 3747 AD-1193443.1 cscsauugCfgAfuuguccagauL96 3388 asUfscudGg(Agn)caaudCgCfaAfuggscsg 3568 UGCCAUUGCGAUUGUCCAGAG 3748 AD-1193444.1 csasuugcgAfUfUfguccagagaL96 3389 usCfsucdTg(G2p)acaaucdGcAfaugsgsc 3569 GCCAUUGCGAUUGUCCAGAGA 3749 AD-1193445.1 asusugcgAfUfUfguccagagauL96 3390 asUfscudCu(G2p)gacaauCfgCfaausgsg 3570 CCAUUGCGAUUGUCCAGAGAC 3750 AD-1193446.1 csasuugcgaUfudGUfccagagacuL96 3391 asdGsucdTc(Tgn)ggacaaUfcdGcaaugsgsc 3571 GCCAUUGCGAUUGUCCAGAGACU 3751 AD-1193447.1 usgscgauUfgUfccagagacuuL96 3392 asAfsgudCu(Cgn)uggadCaAfuCfgcasgsu 3572 AUUGCGAUUGUCCAGAGACUG 3752 AD-1193448.1 gscsgauuGfUfCfcagagacuguL96 3393 asCfsagdTcdTcuggacAfaUfcgcsasa 3573 UUGCGAUUGUCCAGAGACUGG 3753 AD-1193449.1 ususgcgauugUfCfCfagagacuguL96 3394 asCfsagdTcdTcuggacAfaUfcgcaasusg 3574 CAUUGCGAUUGUCCAGAGACUGG 3754 AD-1193450.1 csgsauugUfCfCfagagacugguL96 3395 asCfscadGudCucuggaCfaAfucgscsa 3575 UGCGAUUGUCCAGAGACUGGU 3755 AD-1193451.1 csgsauugUfCfCfagagacugguL96 3396 asCfscadGudCucuggaCfaAfucgscsg 3576 UGCGAUUGUCCAGAGACUGGU 3756 AD-1193452.1 gsasuuguCfCfAfgagacugguuL96 3397 asAfsccdAg(Tgn)cucuggAfcAfaucsgsc 3577 GCGAUUGUCCAGAGACUGGUG 3757 AD-1193452.2 gsasuuguCfCfAfgagacugguuL96 3398 asAfsccdAg(Tgn)cucuggAfcAfaucsgsc 3578 GCGAUUGUCCAGAGACUGGUG 3758 AD-1193453.1 asusugucCfAfGfagacuggugaL96 3399 usCfsacdCadGucucugGfaCfaauscsg 3579 CGAUUGUCCAGAGACUGGUGA 3759 AD-1193454.1 asusugucCfadGagacuggugaL96 3400 usCfsacdCadGucucdTgdGaCfaauscsg 3580 CGAUUGUCCAGAGACUGGUGA 3760 AD-1193455.1 ususguccAfGfAfgacuggugauL96 3401 asUfscadCc(Agn)gucucuGfgAfcaasusc 3581 GAUUGUCCAGAGACUGGUGAC 3761 AD-1193456.1 ususguccAfgAfgacuggugauL96 3402 asUfscadCc(Agn)gucudCudGgAfcaasusc 3582 GAUUGUCCAGAGACUGGUGAC 3762 AD-1193457.1 usgsuccadGadGacuggugacaL96 3403 usdGsucdAc(Cgn)agucdTcUfgdGacasgsu 3583 AUUGUCCAGAGACUGGUGACA 3763 AD-1193458.1 usgsguuuuaUfGfAfaaagcuaggaL96 3404 usCfscudAg(Cgn)uuuucaUfaAfaaccasasc 3584 GUUGGUUUUAUGAAAAGCUAGGA 3764 AD-1193459.1 usgsguuuuaUfgAfaaagcuaggaL96 3405 usCfscudAg(Cgn)uuuudCaUfaAfaaccasgsc 3585 GUUGGUUUUAUGAAAAGCUAGGA 3765 AD-1193460.1 gsgsuuuuaugAfAfaagcuaggaaL96 3406 usUfsccdTa(G2p)cuuuucAfuAfaaaccsgsg 3586 UUGGUUUUAUGAAAAGCUAGGAA 3766 AD-1193461.1 gsusuuuaugAfAfAfagcuaggaauL96 3407 asUfsucdCudAgcuuuuCfaUfaaaacscsa 3587 UGGUUUUAUGAAAAGCUAGGAAG 3767 AD-1193462.1 gsusuuuaugAfAfAfagcuaggaauL96 3408 asUfsucdCudAgcuuuuCfaUfaaaacscsg 3588 UGGUUUUAUGAAAAGCUAGGAAG 3768 AD-1193463.1 ususuuaugaAfAfAfgcuaggaaguL96 3409 asCfsuudCcdTagcuuuUfcAfuaaaascsc 3589 GGUUUUAUGAAAAGCUAGGAAGC 3769 AD-1193464.1 ususuaugaaAfadGCfuaggaagcaL96 3410 usdGscudTc(Cgn)uagcuuUfuCfauaaasgsc 3590 GUUUUAUGAAAAGCUAGGAAGCA 3770 AD-1193465.1 ususaugaaaAfgCfuaggaagcaaL96 3411 usUfsgcdTu(Cgn)cuagdCuUfuUfcauaasgsg 3591 UUUUAUGAAAAGCUAGGAAGCAA 3771 AD-1193466.1 usasugaaaagCfUfaggaagcaauL96 3412 asUfsugdCu(Tgn)ccuagcUfuUfucauasgsg 3592 UUUAUGAAAAGCUAGGAAGCAAC 3772 AD-1193467.1 asusgaaaagCfUfAfggaagcaacuL96 3413 asdGsuudGc(Tgn)uccuagCfuUfuucausgsg 3593 UUAUGAAAAGCUAGGAAGCAACC 3773 AD-1193468.1 usgsaaaagcdTadGgaagcaaccuL96 3414 asdGsgudTg(Cgn)uuccdTadGcUfuuucasusg 3594 UAUGAAAAGCUAGGAAGCAACCU 3774 AD-1193469.1 gsasaaagcudAgdGaagcaaccuuL96 3415 asAfsggdTu(G2p)cuucdCuAfgCfuuuucsgsu 3595 AUGAAAAGCUAGGAAGCAACCUU 3775 AD-519773.3 asasaagcUfaGfGfAfagcaaccuuuL96 3416 asAfsaggUfuGfCfuuccUfaGfcuuuuscsa 3596 UGAAAAGCUAGGAAGCAACCUUU 3776 AD-1193470.1 asasaagcuaGfGfAfagcaaccuuuL96 3417 asAfsagdGudTgcuuccUfaGfcuuuuscsa 3597 UGAAAAGCUAGGAAGCAACCUUU 3777 AD-1193471.1 asasaagcuadGgdAagcaaccuuuL96 3418 asAfsagdGudTgcuudCcUfadGcuuuuscsg 3598 UGAAAAGCUAGGAAGCAACCUUU 3778 AD-1193472.1 asasagcuaggAfAfgcaaccuuuuL96 3419 asAfsaadGg(Tgn)ugcuucCfuAfgcuuususc 3599 GAAAAGCUAGGAAGCAACCUUUC 3779 AD-1193473.1 asasgcuaggAfadGCfaaccuuucuL96 3420 asdGsaadAg(G2p)uugcuuCfcUfagcuususu 3600 AAAAGCUAGGAAGCAACCUUUCG 3780 AD-1193474.1 asgscuaggaAfGfCfaaccuuucguL96 3421 asCfsgadAadGguugcuUfcCfuagcususu 3601 AAAGCUAGGAAGCAACCUUUCGC 3781 AD-1193475.1 csusaggaAfgCfaaccuuucguL96 3422 asCfsgadAadGguugdCuUfcCfuagscsu 3602 AGCUAGGAAGCAACCUUUCGC 3782 AD-1193476.1 gscsuaggaaGfCfAfaccuuucgcuL96 3423 asGfscgdAadAgguugcUfuCfcuagcsusu 3603 AAGCUAGGAAGCAACCUUUCGCC 3783 AD-1193477.1 usasggaagCfAfaccuuucgcuL96 3424 asdGscgdAadAgguugcUfuCfcuasgsc 3604 GCUAGGAAGCAACCUUUCGCC 3784 AD-1193478.1 csusaggaagCfAfAfccuuucgccuL96 3425 asGfsgcdGadAagguugCfuUfccuagscsu 3605 AGCUAGGAAGCAACCUUUCGCCU 3785 AD-1193479.1 asgsgaagCfAfAfccuuucgccuL96 3426 asdGsgcdGadAagguugCfuUfccusgsg 3606 CUAGGAAGCAACCUUUCGCCU 3786 AD-1193480.1 usasggaagcAfAfCfcuuucgccuuL96 3427 asAfsggdCgdAaagguuGfcUfuccuasgsc 3607 GCUAGGAAGCAACCUUUCGCCUG 3787 AD-1193481.1 gsgsaagcAfAfCfcuuucgccuuL96 3428 asAfsggdCgdAaaggdTudGcUfuccsusg 3608 UAGGAAGCAACCUUUCGCCUG 3788 AD-1193482.1 asgsgaagcaAfCfCfuuucgccuguL96 3429 asCfsagdGcdGaaagguUfgCfuuccusasg 3609 CUAGGAAGCAACCUUUCGCCUGU 3789 AD-1193483.1 gsasagcaAfCfCfuuucgccuguL96 3430 asCfsagdGcdGaaagguUfgCfuucscsu 3610 AGGAAGCAACCUUUCGCCUGU 3790 AD-1193484.1 asasgcaaCfCfUfuucgccuguuL96 3431 asAfscadGg(Cgn)gaaaggUfudGcuuscsc 3611 GGAAGCAACCUUUCGCCUGUG 3791 AD-1193485.1 asgscaacCfUfUfucgccuguguL96 3432 asCfsacdAg(G2p)cgaadAgdGuUfgcususc 3612 GAAGCAACCUUUCGCCUGUGC 3792 AD-1193486.1 gscsaaccUfUfUfcgccugugcaL96 3433 usGfscadCa(G2p)gcgadAadGgUfugcsusu 3613 AAGCAACCUUUCGCCUGUGCA 3793

TABLE 51 PNPLA3 Single Dose Screen in Hep3B Cells (% PNPLA3 mRNA remaining) 50 nM 10 nM 1 nM 0.1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-67554.9 32.4 8.8 40.9 10.3 68.4 32.5 100.6 32.3 AD-1193317.1 36.2 5.0 43.8 7.4 47.6 10.5 106.5 28.4 AD-1193318.1 30.9 8.1 42.8 7.0 43.8 5.3 103.5 17.8 AD-1193319.1 22.6 12.2 33.3 7.1 48.8 9.9 63.9 7.5 AD-1193320.1 32.6 11.2 40.8 9.5 50.9 4.1 56.7 26.4 AD-1193321.1 28.1 3.9 27.9 2.0 44.1 8.2 65.1 9.2 AD-1193322.1 22.1 2.3 24.5 2.4 40.9 6.9 36.7 9.3 AD-1193323.1 25.5 5.4 27.2 10.4 39.8 14.1 36.6 15.6 AD-1193324.1 33.7 8.3 44.4 10.0 56.9 10.8 78.0 15.6 AD-1193325.1 75.3 17.9 63.9 3.9 85.6 30.8 101.9 34.7 AD-1193326.1 57.5 13.0 52.4 5.2 61.3 8.9 93.0 16.9 AD-1193327.1 48.8 6.3 45.3 9.9 48.8 10.3 69.8 14.0 AD-1193328.1 58.3 6.7 56.4 5.3 66.9 17.3 68.9 36.3 AD-1193329.1 46.8 7.4 42.2 8.2 55.3 6.3 82.5 8.0 AD-1193330.1 31.2 2.7 34.5 4.1 37.9 3.0 51.4 21.9 AD-1193331.1 54.5 12.4 41.1 6.3 54.9 13.4 58.0 18.2 AD-1193332.1 54.5 23.2 34.6 4.9 58.1 16.4 78.2 28.4 AD-1193333.1 33.7 1.5 42.2 2.2 50.5 3.7 83.9 15.4 AD-1193334.1 71.0 13.1 60.7 6.8 71.4 12.4 82.8 14.3 AD-1193335.1 61.4 13.8 56.9 4.5 60.3 6.3 78.7 21.2 AD-1193336.1 53.8 3.0 58.5 13.9 64.4 5.7 78.6 28.1 AD-519346.3 24.2 4.9 36.1 11.5 36.6 6.2 82.0 24.6 AD-1193337.1 18.2 3.6 22.6 3.8 43.5 19.6 39.8 9.5 AD-1193338.1 28.6 3.8 36.7 7.0 42.4 2.0 86.3 20.2 AD-1193339.1 25.9 3.6 31.4 3.6 45.5 9.0 78.7 15.8 AD-1193340.1 33.8 2.7 35.2 4.7 42.4 11.5 70.2 13.0 AD-1193341.1 28.6 5.5 34.0 2.4 57.0 10.4 63.2 19.3 AD-1193342.1 25.7 4.4 31.1 6.8 39.0 4.1 66.3 11.2 AD-1193343.1 24.6 5.9 32.5 2.5 37.8 4.9 81.0 16.1 AD-1193344.1 23.0 3.3 34.6 4.2 33.2 7.9 46.7 12.8 AD-1193345.1 23.4 4.2 35.3 11.8 40.6 9.6 79.2 10.7 AD-1193346.1 18.7 2.9 30.6 3.4 47.9 4.7 90.3 24.4 AD-1193347.1 28.5 6.6 43.9 3.9 57.9 9.7 73.1 21.1 AD-1193348.1 38.2 2.4 38.9 2.8 52.3 9.7 64.7 15.0 AD-1193349.1 26.4 3.9 40.9 7.1 43.5 4.6 75.6 4.4 AD-519347.4 24.5 2.7 33.1 9.0 48.9 15.5 79.8 27.0 AD-1193350.1 20.8 0.9 31.2 7.1 42.3 11.3 61.4 8.3 AD-1193351.1 19.2 3.4 31.3 3.1 40.2 5.4 58.2 24.1 AD-1193352.1 21.4 3.8 38.4 11.3 37.2 5.0 83.9 5.8 AD-1193353.1 26.3 4.3 36.5 5.3 55.2 10.8 96.6 16.1 AD-1193354.1 27.3 6.9 35.9 1.4 52.9 3.6 75.3 4.3 AD-1193355.1 37.3 4.4 43.7 9.4 57.4 7.0 86.3 12.1 AD-1193356.1 38.8 5.7 48.4 5.0 60.5 11.2 87.4 4.9 AD-1193357.1 37.2 4.5 49.0 11.9 59.1 17.2 84.1 8.9 AD-1193358.1 34.9 9.9 45.9 9.9 56.5 9.7 57.3 16.4 AD-519350.4 32.6 3.5 36.3 13.8 47.7 9.4 96.7 23.3 AD-1193359.1 33.5 2.0 33.5 1.6 54.4 6.9 85.0 18.8 AD-1193360.1 28.7 5.5 39.8 3.3 50.9 9.7 85.3 14.3 AD-1193361.1 33.0 4.4 42.5 4.3 49.0 4.3 59.5 6.2 AD-1193362.1 33.3 6.6 46.4 10.6 62.4 12.3 61.4 7.8 AD-1193363.1 25.4 2.7 35.3 4.8 46.1 6.1 59.5 21.1 AD-1193364.1 28.7 3.3 29.0 3.4 53.3 14.5 67.2 13.3 AD-1193365.1 20.7 3.8 29.3 9.2 40.6 4.3 56.7 13.2 AD-1193366.1 30.5 1.7 39.8 7.1 41.7 6.3 82.0 30.9 AD-1193367.1 29.3 9.0 37.5 10.3 41.9 7.7 77.8 26.0 AD-1193368.1 36.2 7.7 42.7 6.1 52.5 9.8 72.3 16.8 AD-1193369.1 51.7 23.9 47.4 4.8 45.9 3.4 70.7 15.6 AD-1193370.1 29.9 4.6 43.4 2.9 52.7 14.2 76.6 20.3 AD-1193371.1 34.5 5.1 31.4 7.4 38.9 6.5 65.9 11.0 AD-1193372.1 30.8 3.7 39.4 8.7 48.0 6.4 77.3 5.3 AD-519757.4 32.3 1.1 58.0 8.1 61.1 8.0 83.2 15.0 AD-1193373.1 40.8 6.6 59.6 12.1 65.9 9.1 99.6 27.0 AD-1193374.1 55.5 4.7 61.5 8.3 78.6 11.7 97.8 22.6 AD-1193375.1 65.6 11.1 74.6 14.7 94.6 15.4 89.3 27.0 AD-1193376.1 65.7 3.1 78.8 11.1 94.1 19.5 85.4 16.2 AD-1193377.1 53.4 5.9 64.0 17.9 72.2 16.5 94.9 10.3 AD-1193378.1 53.3 6.9 72.6 8.3 72.1 10.2 70.6 25.9 AD-1193379.1 36.9 7.7 49.7 11.0 59.0 12.5 85.8 10.1 AD-1193380.1 40.9 9.3 61.5 19.2 71.9 12.0 101.4 13.9 AD-1193381.1 55.7 15.8 69.3 6.2 90.6 38.1 106.0 12.6 AD-1193382.1 78.9 14.3 96.4 13.8 94.3 20.3 130.7 11.3 AD-1193383.1 76.4 23.2 87.0 24.5 90.4 9.8 116.3 14.5 AD-1193384.1 107.4 7.9 97.1 13.0 104.3 17.4 140.3 15.9 AD-1193385.1 46.1 10.1 72.3 21.2 54.1 2.4 84.5 20.3 AD-1193386.1 53.4 9.5 64.9 10.2 79.4 7.1 102.8 12.3 AD-1193387.1 58.3 24.2 54.3 3.6 77.1 20.3 93.3 15.4 AD-1193388.1 42.9 4.4 63.4 8.0 89.4 7.3 109.9 33.5 AD-1193389.1 70.9 13.7 88.8 13.4 97.8 15.5 119.3 30.2 AD-1193390.1 62.6 14.5 70.4 7.1 100.9 35.3 120.1 21.6 AD-1193391.1 73.6 8.9 85.4 23.5 115.5 35.0 120.4 18.2 AD-520018.9 25.3 4.0 41.2 5.9 60.8 9.8 110.1 16.8 AD-1193392.1 27.9 6.9 42.9 11.9 51.1 17.6 85.4 10.3 AD-1193393.1 34.4 10.9 39.7 14.6 47.3 11.7 78.6 12.3 AD-1193394.1 33.8 6.8 52.5 16.4 56.8 4.6 62.7 23.2 AD-1193395.1 27.7 9.0 42.8 10.1 54.3 7.6 69.2 16.8 AD-1193396.1 36.3 11.7 51.3 3.4 58.4 18.1 90.3 23.6 AD-1193397.1 44.5 13.8 62.3 12.7 50.0 18.9 74.2 16.6 AD-1193398.1 27.3 7.4 47.5 5.1 52.8 4.0 87.0 19.6 AD-1193399.1 35.8 6.6 45.3 10.1 58.4 15.9 89.7 11.7 AD-1193400.1 27.1 5.8 47.1 14.5 59.1 13.8 72.1 16.1 AD-1193401.1 26.7 17.3 46.4 2.6 64.4 8.5 85.5 22.4 AD-1193402.1 25.9 14.1 38.7 12.2 48.1 3.9 64.4 22.7 AD-1193403.1 30.4 8.6 44.7 7.2 54.5 4.0 81.1 15.1 AD-1193404.1 27.6 12.1 46.4 4.7 36.0 13.6 76.3 18.6 AD-1193405.1 26.8 5.4 38.3 3.6 51.1 3.1 76.2 10.8 AD-1193406.1 29.0 9.9 35.3 6.9 54.2 3.3 96.3 21.3 AD-1193407.1 21.5 7.1 32.0 7.3 47.7 5.0 61.4 11.1 AD-520053.5 11.3 6.6 23.9 5.3 30.9 8.0 49.2 14.7 AD-1193408.1 24.3 7.6 40.1 1.4 60.8 12.1 83.1 22.4 AD-1193409.1 21.1 6.9 26.7 4.8 51.4 15.8 76.5 9.6 AD-1193410.1 20.4 3.3 24.1 4.4 33.5 3.9 91.0 10.4 AD-1193411.1 23.3 12.5 28.3 7.0 48.4 6.2 83.1 30.9 AD-1193412.1 21.0 7.6 28.0 2.5 43.4 8.3 71.1 14.0 AD-1193413.1 28.6 5.1 23.2 1.6 45.1 8.0 65.8 4.7 AD-1193414.1 23.7 8.6 28.7 11.4 40.0 6.1 65.9 23.7 AD-1193415.1 11.1 3.3 30.7 5.2 38.1 9.0 58.4 11.2 AD-1193416.1 32.8 6.3 38.5 5.4 47.7 12.0 64.9 9.2 AD-1193417.1 29.1 3.7 43.6 12.4 58.3 4.0 87.4 15.6 AD-1193418.1 46.2 11.5 31.7 5.5 56.1 10.5 80.3 11.9 AD-1193419.1 29.7 2.2 42.7 5.6 59.5 11.8 79.1 9.3 AD-1193420.1 29.3 3.2 29.0 7.4 38.1 4.2 70.6 15.2 AD-1193421.1 18.7 6.7 25.6 9.7 29.1 8.7 47.1 7.6 AD-1193422.1 25.6 5.7 48.1 8.4 56.9 22.0 58.7 11.9 AD-1193423.1 55.6 8.9 68.1 6.7 78.2 3.1 77.5 12.5 AD-1193424.1 39.2 5.0 50.4 10.8 66.3 15.0 100.3 28.7 AD-1193425.1 58.7 9.2 54.7 5.1 69.9 12.0 88.5 17.5 AD-1193426.1 38.0 3.3 41.3 5.0 56.7 8.6 79.3 2.5 AD-1193427.1 41.7 7.8 37.6 6.0 60.0 5.3 72.0 4.0 AD-1193428.1 38.0 4.3 32.2 13.9 43.1 19.7 56.7 22.0 AD-1193429.1 40.2 10.5 43.4 13.4 50.0 11.4 52.3 13.4 AD-1193430.1 47.3 6.8 54.1 7.1 74.4 13.3 92.8 7.3 AD-1193431.1 45.8 7.9 53.6 7.7 71.8 7.3 116.6 14.1 AD-1193432.1 57.3 3.6 52.2 13.6 83.6 3.8 99.5 12.6 AD-1193433.1 35.2 13.7 47.6 14.8 73.4 22.0 71.7 7.4 AD-1193434.1 75.2 8.4 95.7 19.4 107.3 18.0 105.9 27.1 AD-1193435.1 75.3 7.5 63.1 10.9 79.2 18.9 70.4 20.0 AD-1193436.1 37.1 7.2 42.6 12.0 65.7 22.3 79.1 20.0 AD-1193437.1 26.3 7.3 76.6 41.5 50.4 16.0 61.8 14.3 AD-1193438.1 45.5 8.4 51.1 3.0 71.2 3.6 92.0 7.5 AD-1193439.1 60.8 6.5 66.7 4.4 91.7 8.3 125.9 18.1 AD-1193440.1 128.1 12.3 86.7 11.2 99.9 8.2 83.7 19.6 AD-805631.3 76.1 9.0 73.7 7.9 109.0 18.9 100.3 15.6 AD-1193441.1 71.4 12.6 91.6 13.0 116.1 20.7 103.1 26.0 AD-1193442.1 51.4 6.5 54.7 5.6 67.8 15.5 106.9 31.6 AD-1193443.1 31.6 8.6 41.7 11.1 44.0 12.5 79.0 29.5 AD-1193444.1 83.4 2.4 73.6 17.1 92.4 11.7 84.3 25.3 AD-1193445.1 86.5 4.5 85.5 15.1 94.5 27.8 103.3 9.7 AD-1193446.1 79.6 15.2 72.5 9.0 91.1 18.9 87.7 10.1 AD-1193447.1 117.4 18.9 99.1 15.4 115.7 27.8 91.8 20.5 AD-1193448.1 47.7 7.7 52.1 7.4 74.2 16.2 93.2 24.0 AD-1193449.1 43.0 12.3 52.4 10.0 64.4 1.9 84.5 10.8 AD-1193450.1 43.2 14.8 68.7 11.6 71.6 16.2 68.3 12.5 AD-1193451.1 52.4 13.9 72.2 6.5 77.8 15.9 63.2 10.4 AD-1193452.1 70.9 12.6 81.1 5.6 97.2 19.5 97.2 11.8 AD-1193452.2 71.9 14.7 89.6 13.0 112.4 14.3 109.8 22.8 AD-1193453.1 88.9 15.4 83.1 11.0 97.1 27.3 98.8 12.3 AD-1193454.1 119.8 17.1 96.2 18.7 120.9 12.9 112.1 16.2 AD-1193455.1 106.1 14.5 93.4 23.8 116.1 10.2 103.7 24.9 AD-1193456.1 85.1 22.7 86.9 10.6 97.3 25.7 102.0 11.2 AD-1193457.1 53.2 23.9 61.2 22.6 82.8 12.8 90.0 14.0 AD-1193458.1 46.2 33.5 76.4 16.5 82.2 15.3 76.4 11.7 AD-1193459.1 68.2 12.4 79.6 21.7 95.4 11.0 104.3 8.7 AD-1193460.1 58.1 14.5 69.8 10.3 95.6 12.7 85.6 20.4 AD-1193461.1 68.4 9.2 61.0 9.3 75.1 13.2 77.2 5.0 AD-1193462.1 46.4 9.0 56.5 10.5 74.9 13.1 78.3 14.0 AD-1193463.1 62.5 14.0 66.1 7.6 92.5 11.0 98.7 23.3 AD-1193464.1 52.2 13.4 49.0 10.1 50.8 15.8 54.5 18.4 AD-1193465.1 41.9 15.2 64.6 7.4 48.6 16.7 64.3 11.9 AD-1193466.1 53.0 7.2 55.1 9.1 75.8 12.0 77.0 24.4 AD-1193467.1 57.0 14.0 54.3 7.4 73.7 6.1 110.5 30.2 AD-1193468.1 86.8 13.8 94.0 14.8 98.8 9.8 108.8 19.6 AD-1193469.1 90.2 12.4 91.8 6.9 100.2 15.6 113.3 27.2 AD-519773.3 56.2 2.7 57.4 13.3 54.7 9.0 107.0 5.6 AD-1193470.1 55.4 8.7 39.1 7.8 42.3 9.2 68.4 10.3 AD-1193471.1 46.6 21.6 48.9 5.9 36.5 4.8 60.4 19.0 AD-1193472.1 70.7 22.1 76.2 10.3 101.6 15.8 63.0 1.2 AD-1193473.1 62.6 16.1 60.0 16.7 82.6 6.0 107.0 23.5 AD-1193474.1 55.0 15.6 57.0 12.2 70.5 15.9 81.5 14.3 AD-1193475.1 71.9 4.8 59.2 2.5 74.5 13.3 116.9 22.5 AD-1193476.1 67.6 12.1 65.6 6.0 76.1 6.5 132.8 18.8 AD-1193477.1 67.6 3.5 65.7 13.6 62.6 7.6 108.6 28.3 AD-1193478.1 65.9 18.6 50.6 5.2 49.8 8.2 91.1 16.4 AD-1193479.1 34.1 12.3 45.2 4.2 39.2 11.6 106.3 37.4 AD-1193480.1 30.1 8.0 52.1 11.6 51.3 5.7 83.1 14.8 AD-1193481.1 33.2 4.9 41.1 7.0 67.2 15.6 96.1 21.9 AD-1193482.1 38.8 5.8 52.8 13.4 57.2 6.8 99.4 26.1 AD-1193483.1 42.5 11.4 60.0 21.7 77.5 14.5 112.1 35.1 AD-1193484.1 47.2 6.9 48.9 7.7 80.6 6.7 79.2 16.9 AD-1193485.1 65.2 5.0 78.8 10.8 82.5 10.8 94.5 16.5 AD-1193486.1 94.3 12.1 93.6 10.7 71.4 30.8 106.2 9.5

TABLE 52 PNPLA3 Single Dose Screen (Free Uptake) in Primary Cynomolgus Hepatocytes (PCH) (% PNPLA3 mRNA remaining) 500 nM 100 nM 10 nM 1 nM Duplex Avg SD Avg SD Avg SD Avg SD AD-67554.9 54.4 n/a 38.5 21.8 50.1 6.1 108.8 100.6 AD-1193317.1 48.4 16.9 75.0 8.8 67.4 24.1 91.0 31.5 AD-1193318.1 55.5 14.8 58.5 54.9 51.7 19.9 98.1 19.1 AD-1193319.1 39.2 9.0 66.2 7.1 76.1 31.6 87.4 23.7 AD-1193320.1 64.8 7.0 52.4 24.2 79.4 1.2 110.3 38.3 AD-1193321.1 51.1 21.2 52.4 35.8 64.5 20.2 78.8 29.1 AD-1193322.1 52.6 21.1 43.0 25.8 75.4 19.2 119.5 9.9 AD-1193323.1 58.5 33.4 39.1 18.7 63.7 29.6 53.8 9.2 AD-1193324.1 54.6 6.9 47.4 25.4 63.6 11.5 70.1 18.0 AD-1193325.1 81.0 18.7 104.1 19.3 110.8 8.1 97.8 19.1 AD-1193326.1 57.7 5.5 79.2 13.0 85.4 6.9 93.8 14.6 AD-1193327.1 81.2 33.7 81.9 1.5 96.2 21.0 92.6 9.1 AD-1193328.1 72.5 13.7 104.9 8.6 76.8 6.6 86.6 11.1 AD-1193329.1 74.1 6.9 109.9 21.1 91.1 11.3 100.0 21.9 AD-1193330.1 70.4 39.4 109.0 32.8 84.8 9.0 113.5 26.9 AD-1193331.1 85.6 31.9 82.9 34.6 71.1 19.3 129.4 38.7 AD-1193332.1 43.7 2.4 60.0 26.9 58.0 11.5 60.9 21.4 AD-1193333.1 52.7 15.8 77.7 5.7 85.6 7.9 72.4 16.0 AD-1193334.1 76.0 15.1 87.3 3.7 95.1 17.8 88.5 18.0 AD-1193335.1 66.4 11.9 84.4 8.0 81.4 12.6 83.1 9.1 AD-1193336.1 68.2 19.1 91.5 14.9 82.0 12.5 79.6 23.7 AD-519346.3 64.0 25.2 85.5 11.1 91.2 25.5 85.5 14.0 AD-1193337.1 38.0 4.8 72.8 28.5 78.0 18.5 90.7 1.8 AD-1193338.1 47.0 4.8 76.9 35.9 61.0 16.6 60.9 16.7 AD-1193339.1 54.1 20.8 60.2 11.0 86.7 23.9 80.9 16.3 AD-1193340.1 55.2 20.6 65.7 16.6 107.2 55.6 78.7 13.2 AD-1193341.1 45.4 9.6 55.8 3.2 78.0 5.9 78.2 8.1 AD-1193342.1 41.4 10.0 62.7 7.7 76.7 10.8 89.2 5.1 AD-1193343.1 45.6 12.4 71.8 8.6 68.6 8.3 88.4 6.0 AD-1193344.1 55.8 7.6 58.3 18.6 73.8 3.6 66.9 20.4 AD-1193345.1 62.1 3.6 74.5 36.7 78.6 36.5 58.7 19.9 AD-1193346.1 54.5 5.0 55.0 3.8 97.5 5.4 85.3 11.0 AD-1193347.1 66.3 19.3 67.3 5.6 107.0 29.0 95.2 9.4 AD-1193348.1 65.0 15.8 80.9 13.5 99.3 26.6 98.7 22.8 AD-1193349.1 50.3 14.0 70.3 22.0 91.5 11.7 85.3 10.7 AD-519347.4 76.6 24.3 55.9 8.7 91.0 14.8 88.7 10.3 AD-1193350.1 56.8 28.0 55.4 2.2 77.3 10.2 84.2 13.1 AD-1193351.1 47.1 5.1 82.5 11.7 89.6 17.1 93.2 9.6 AD-1193352.1 54.8 4.3 58.3 6.1 67.4 27.9 61.7 0.9 AD-1193353.1 58.3 15.9 59.3 9.9 98.3 8.7 81.2 14.2 AD-1193354.1 56.4 18.6 55.8 12.0 86.1 8.3 81.4 10.0 AD-1193355.1 94.6 28.6 85.5 21.2 93.8 5.1 92.3 16.1 AD-1193356.1 47.3 5.5 67.9 1.3 95.3 7.7 118.7 73.9 AD-1193357.1 77.6 29.8 71.4 4.8 105.7 13.6 100.2 18.9 AD-1193358.1 58.6 4.9 64.3 5.2 80.3 16.9 90.7 10.9 AD-519350.4 60.4 15.2 59.5 23.5 57.5 3.4 60.3 21.1 AD-1193359.1 80.7 10.4 96.2 7.6 112.2 13.5 91.1 16.0 AD-1193360.1 102.6 27.0 93.5 14.4 114.6 11.9 81.5 9.4 AD-1193361.1 82.2 14.8 87.1 n/a 115.4 19.7 165.0 127.4 AD-1193362.1 109.2 38.9 97.3 11.6 109.6 9.1 149.9 76.0 AD-1193363.1 77.0 20.4 91.1 5.8 90.6 8.1 90.0 5.6 AD-1193364.1 68.6 17.3 96.6 10.5 97.0 9.0 100.3 15.4 AD-1193365.1 77.6 29.1 96.0 13.2 100.9 5.3 90.6 9.3 AD-1193366.1 68.9 9.1 87.9 15.9 83.7 20.1 51.9 18.7 AD-1193367.1 75.7 15.2 96.0 16.5 112.8 9.9 74.0 4.5 AD-1193368.1 86.7 14.7 90.9 14.5 115.5 20.4 82.2 8.8 AD-1193369.1 72.4 26.0 101.3 5.4 142.9 32.6 125.0 45.8 AD-1193370.1 94.2 29.8 101.4 7.7 121.2 6.5 95.5 17.0 AD-1193371.1 72.8 21.4 90.5 5.3 113.5 10.1 87.6 8.0 AD-1193372.1 109.7 37.6 102.7 19.0 107.2 20.6 84.2 10.4 AD-519757.4 66.8 35.1 108.2 8.5 97.9 26.8 93.8 9.6 AD-1193373.1 53.5 7.3 75.7 20.4 74.5 29.8 102.6 55.7 AD-1193374.1 94.6 35.4 98.0 6.7 121.0 23.6 84.3 6.0 AD-1193375.1 89.8 20.3 103.1 3.3 122.8 8.5 89.2 24.7 AD-1193376.1 95.7 29.8 126.7 28.8 105.9 17.9 72.3 8.8 AD-1193377.1 61.4 9.9 116.9 20.0 105.4 14.0 78.6 11.8 AD-1193378.1 68.4 21.1 102.5 12.8 109.7 15.9 87.4 5.4 AD-1193379.1 57.7 13.8 99.3 27.4 99.5 32.7 86.4 1.7 AD-1193380.1 77.9 32.2 78.4 19.1 83.8 17.7 65.5 3.2 AD-1193381.1 71.1 9.8 109.6 19.8 122.9 25.7 135.4 52.7 AD-1193382.1 101.0 15.9 126.4 24.8 128.1 20.3 88.1 10.8 AD-1193383.1 91.0 19.6 114.0 13.5 107.7 13.0 87.5 14.8 AD-1193384.1 93.8 7.0 118.1 21.9 106.8 13.5 202.7 185.4 AD-1193385.1 70.8 17.3 92.1 9.2 109.5 6.6 103.7 9.0 AD-1193386.1 65.9 22.5 138.5 36.6 109.7 32.7 135.6 31.6 AD-1193387.1 46.6 3.6 83.9 36.6 64.8 15.3 230.5 271.8 AD-1193388.1 68.2 14.7 99.9 22.9 104.4 22.7 89.4 n/a AD-1193389.1 72.9 9.2 120.7 23.0 106.3 8.8 n/a n/a AD-1193390.1 80.8 10.3 94.7 9.7 111.4 9.8 117.2 n/a AD-1193391.1 93.0 29.3 117.7 7.0 108.9 14.0 n/a n/a AD-520018.9 75.1 35.9 85.7 11.8 86.3 2.5 n/a n/a AD-1193392.1 95.1 34.0 117.4 11.4 115.5 6.3 292.6 n/a AD-1193393.1 57.7 28.7 148.4 13.8 104.6 38.8 121.0 44.6 AD-1193394.1 71.1 42.0 68.9 15.8 70.7 21.2 53.3 11.3 AD-1193395.1 79.1 22.9 91.2 27.2 84.3 9.7 164.8 152.7 AD-1193396.1 60.4 17.7 104.3 15.3 95.5 21.0 159.6 149.5 AD-1193397.1 106.2 42.2 89.2 26.0 90.7 20.4 92.0 n/a AD-1193398.1 51.6 9.8 86.4 5.0 74.8 15.4 93.7 30.5 AD-1193399.1 94.0 19.4 100.9 27.6 88.2 28.8 114.5 7.3 AD-1193400.1 67.4 23.0 108.8 24.8 81.0 23.7 141.2 31.6 AD-1193401.1 58.2 46.0 104.3 n/a 95.3 33.1 51.2 14.7 AD-1193402.1 85.6 16.7 87.5 7.3 93.8 27.4 84.8 25.2 AD-1193403.1 85.6 5.7 78.6 10.2 93.4 31.2 81.0 10.0 AD-1193404.1 77.2 23.3 86.6 21.8 86.2 29.7 76.6 17.2 AD-1193405.1 64.6 14.4 83.3 4.5 104.0 40.2 95.9 24.6 AD-1193406.1 91.7 18.0 89.9 30.4 112.1 20.8 68.2 27.3 AD-1193407.1 98.2 43.4 109.1 62.9 144.8 1.4 54.1 19.2 AD-520053.5 66.3 18.3 88.0 41.5 103.2 44.9 56.0 15.6 AD-1193408.1 47.2 2.1 41.0 12.2 84.0 21.0 63.0 23.6 AD-1193409.1 64.7 10.7 59.4 8.3 76.3 9.7 95.1 5.2 AD-1193410.1 62.9 10.1 48.1 3.2 72.6 7.7 104.7 20.2 AD-1193411.1 53.0 5.2 48.3 3.5 70.9 14.0 95.5 11.5 AD-1193412.1 48.9 2.2 43.6 4.3 69.7 9.3 91.1 6.6 AD-1193413.1 58.6 4.9 55.0 8.9 84.6 5.4 100.2 11.6 AD-1193414.1 82.3 15.4 76.3 16.6 124.2 13.5 105.5 27.3 AD-1193415.1 80.8 14.3 111.6 10.4 120.4 34.2 84.8 5.2 AD-1193416.1 44.3 17.6 51.8 21.1 63.4 3.7 78.7 19.1 AD-1193417.1 54.6 19.1 51.2 4.5 65.4 6.7 82.0 5.4 AD-1193418.1 64.5 7.1 62.9 3.4 65.9 3.0 93.2 2.6 AD-1193419.1 55.5 3.0 51.8 3.6 67.3 1.4 77.9 13.2 AD-1193420.1 54.5 3.8 51.7 3.7 79.7 11.6 89.0 9.3 AD-1193421.1 63.3 4.7 62.3 4.1 101.2 8.2 80.0 10.4 AD-1193422.1 137.5 11.1 106.8 11.5 122.0 39.4 85.9 13.3 AD-1193423.1 63.3 37.2 79.9 2.0 110.5 47.4 102.9 13.4 AD-1193424.1 66.9 9.9 59.7 5.7 74.1 4.8 101.5 8.0 AD-1193425.1 80.4 8.1 78.9 13.3 73.7 8.3 94.9 4.0 AD-1193426.1 53.1 4.4 55.7 3.4 71.8 14.5 68.5 4.5 AD-1193427.1 71.1 5.7 81.1 14.3 81.3 11.3 79.5 10.2 AD-1193428.1 76.2 4.7 63.5 4.0 113.3 30.1 83.7 10.3 AD-1193429.1 101.4 3.2 116.4 47.0 160.6 n/a 89.7 28.5 AD-1193430.1 45.8 38.4 53.8 15.8 83.4 16.3 113.0 1.1 AD-1193431.1 63.6 4.2 65.2 6.5 72.9 1.8 113.3 9.6 AD-1193432.1 68.0 7.5 71.2 2.0 75.8 7.3 111.8 10.1 AD-1193433.1 71.7 5.2 70.2 8.4 70.6 2.3 106.4 11.2 AD-1193434.1 81.4 11.3 77.2 4.6 79.3 4.0 93.1 7.0 AD-1193435.1 87.4 8.7 87.6 6.8 94.9 22.5 90.5 12.3 AD-1193436.1 71.3 3.1 65.7 7.0 105.7 4.6 91.1 10.6 AD-1193437.1 77.3 5.3 72.6 11.3 141.9 9.7 95.0 15.0 AD-1193438.1 41.1 18.3 33.7 32.0 81.9 21.3 97.1 17.2 AD-1193439.1 65.3 5.1 69.9 1.3 70.4 8.2 127.4 20.2 AD-1193440.1 104.4 13.4 99.9 21.5 84.6 5.0 97.7 20.0 AD-805631.3 79.1 11.9 72.1 5.5 80.3 4.2 113.0 23.4 AD-1193441.1 86.8 4.5 83.1 5.8 97.6 43.6 100.0 4.8 AD-1193442.1 80.2 8.9 72.2 18.5 100.2 13.0 106.3 10.8 AD-1193443.1 78.1 6.5 82.3 13.4 128.2 33.5 95.1 12.7 AD-1193444.1 64.0 25.7 68.3 32.7 65.0 1.0 96.2 16.6 AD-1193445.1 89.9 3.1 82.1 8.0 83.9 6.2 122.6 17.3 AD-1193446.1 91.0 3.6 72.2 26.3 84.8 18.6 106.8 13.6 AD-1193447.1 89.7 13.6 90.9 4.7 89.7 17.9 120.1 8.8 AD-1193448.1 63.6 2.9 68.7 7.1 79.0 4.4 99.2 12.5 AD-1193449.1 74.6 3.9 70.3 6.9 95.2 15.3 96.7 13.9 AD-1193450.1 81.5 5.8 80.2 4.9 169.5 25.9 98.5 16.1 AD-1193451.1 95.7 26.0 82.5 8.3 216.4 n/a 106.5 5.6 AD-1193452.1 66.7 24.1 49.9 40.7 74.7 1.1 81.5 24.5 AD-1193452.2 87.1 6.8 88.1 8.1 90.0 13.8 137.9 18.9 AD-1193453.1 82.9 9.0 82.9 2.9 81.5 10.7 141.1 11.7 AD-1193454.1 87.6 23.1 88.8 3.9 101.0 19.5 111.0 17.2 AD-1193455.1 102.3 32.6 92.3 13.5 87.1 10.5 91.3 7.0 AD-1193456.1 96.7 14.6 87.2 5.4 105.9 12.1 115.5 10.1 AD-1193457.1 85.5 9.2 85.9 8.6 170.3 23.2 113.7 5.8 AD-1193458.1 75.7 17.2 91.9 22.6 142.2 22.9 90.9 2.9 AD-1193459.1 70.5 31.2 69.6 18.7 65.9 19.3 95.5 32.7 AD-1193460.1 83.5 4.8 77.5 9.3 75.2 0.5 121.8 17.7 AD-1193461.1 64.3 7.6 67.3 10.5 70.8 5.6 115.5 16.9 AD-1193462.1 62.9 1.3 61.4 4.1 81.9 8.5 109.8 15.6 AD-1193463.1 74.5 10.8 68.4 3.4 107.4 6.8 97.8 19.2 AD-1193464.1 77.3 5.6 78.5 11.5 147.0 26.5 105.9 13.6 AD-1193465.1 86.4 36.5 83.0 13.2 193.7 n/a 89.4 17.2 AD-1193466.1 51.6 12.2 48.2 10.9 68.8 12.4 91.0 26.1 AD-1193467.1 99.4 18.0 84.9 3.2 74.5 6.4 126.9 4.0 AD-1193468.1 97.3 13.6 97.0 12.8 88.4 8.6 123.7 14.9 AD-1193469.1 89.6 7.6 78.1 17.0 99.4 13.3 101.6 16.1 AD-519773.3 63.3 4.0 60.5 7.6 112.7 24.3 129.6 23.3 AD-1193470.1 70.9 9.0 64.6 4.3 135.9 24.9 106.4 23.0 AD-1193471.1 77.9 23.5 85.3 2.5 161.9 11.1 80.6 8.5 AD-1193472.1 63.9 39.9 64.2 26.0 77.2 15.5 74.3 17.8 AD-1193473.1 86.6 5.0 80.5 3.9 79.6 10.8 115.1 7.9 AD-1193474.1 90.1 2.3 81.1 4.6 79.5 5.5 124.9 14.6 AD-1193475.1 82.5 22.7 84.1 8.0 79.1 8.1 118.2 19.9 AD-1193476.1 86.6 5.2 85.2 8.8 83.2 3.5 122.2 9.6 AD-1193477.1 81.2 1.1 80.2 8.8 87.0 9.1 101.2 8.3 AD-1193478.1 100.9 16.7 85.4 5.6 155.3 20.6 109.3 6.3 AD-1193479.1 119.3 1.8 90.0 41.1 171.3 27.4 75.2 15.7 AD-1193480.1 46.4 25.9 62.2 19.4 57.8 12.2 72.1 18.6 AD-1193481.1 65.0 48.7 72.5 24.4 83.0 6.9 105.4 12.0 AD-1193482.1 76.5 22.8 74.0 16.5 86.0 18.6 108.5 9.9 AD-1193483.1 82.1 21.3 68.0 34.6 79.0 4.3 76.6 17.6 AD-1193484.1 85.8 38.5 63.2 38.2 81.8 11.2 83.2 18.2 AD-1193485.1 75.7 11.8 81.3 10.4 99.7 14.4 71.1 22.4 AD-1193486.1 106.3 27.5 86.8 11.7 172.4 64.3 77.1 28.3

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of nucleotides 1214-1234 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of nucleotides 1572-1592 of SEQ ID NO:2, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein both the sense strand and the antisense strand independently comprise at least one phosphorothioate or methylphosphonate internucleotide linkage, and wherein at least one strand is conjugated to a ligand.
 2. The dsRNA agent of claim 1, wherein the sense strand comprises 2-6 2′-fluoro modified nucleotides.
 3. The dsRNA agent of claim 2, wherein the sense strand comprises 4 2′-fluoro modified nucleotides.
 4. The dsRNA agent of claim 1, wherein the antisense strand comprises 2-8 2′-fluoro modified nucleotides.
 5. The dsRNA agent of claim 4, wherein the antisense strand comprises 6 2′-fluoro modified nucleotides.
 6. The dsRNA agent of claim 1, wherein the sense strand comprises 4 2′-fluoro modified nucleotides at nucleotides 7 and 9-11, counting from the 5′-end, and the antisense strand comprises 6 2′-fluoro modified nucleotides at nucleotides 2, 6, 8, 9, 14 and 16, counting from the 5′-end.
 7. The dsRNA agent of claim 1, wherein the sense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at the 5′-terminus.
 8. The dsRNA agent of claim 1, wherein the antisense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at both the 5′- and the 3′-terminus.
 9. The dsRNA agent of claim 1, wherein the sense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at the 5′-terminus and the antisense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at both the 5′- and the 3′-terminus.
 10. The dsRNA agent of claim 1, wherein the ligand is conjugated to the 3′-end of the sense strand.
 11. The dsRNA agent of claim 1, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
 12. The dsRNA agent of claim 11, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.
 13. The dsRNA agent of claim 12, wherein the ligand is


14. The dsRNA agent of claim 13, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O.
 15. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand differs by no more than 3 modified nucleotides from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 3 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage.
 16. The dsRNA agent of claim 15, wherein the sense strand differs by no more than 2 modified nucleotides from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 2 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732).
 17. The dsRNA agent of claim 15, wherein the sense strand differs by no more than 1 modified nucleotide from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 1 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732).
 18. The dsRNA agent of claim 15, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O.
 19. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand comprises the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage.
 20. The dsRNA agent of claim 19, wherein the sense strand comprises the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuauguL96-3′ (SEQ ID NO:2648) and wherein the antisense strand comprises the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; s is a phosphorothioate linkage, and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
 21. The dsRNA agent of claim 19, wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic:

wherein X is O.
 22. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand consists of the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand consists of the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage; and wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic:

wherein X is O.
 23. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim
 1. 24. The pharmaceutical composition of claim 23, wherein dsRNA agent is in an unbuffered solution.
 25. The pharmaceutical composition of claim 24, wherein the unbuffered solution is saline or water.
 26. The pharmaceutical composition of claim 23, wherein said dsRNA agent is in a buffer solution.
 27. The pharmaceutical composition of claim 26, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
 28. The pharmaceutical composition of claim 27, wherein the buffer solution is phosphate buffered saline (PBS).
 29. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim
 21. 30. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim
 22. 